Momp telonanoparticles, and related compositions, methods and systems

ABSTRACT

A telodendrimer-nanolipoprotein particle (t-NLP), comprising one or more membrane forming lipids, one or more telodendrimers, and a scaffold protein and a Chlamydia major outer membrane protein (MOMP) comprising a MOMP hydrophobic region, and related compositions methods and systems.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/500,435, entitled “MOMP telonanoparticles, and relatedcompositions, methods and systems” filed on May 2, 2017 with docketnumber IL-13105, the content of which is incorporated herein byreference in its entirety.

STATEMENT OF GOVERNMENT GRANT

The invention was made with Government support under Contract No.DE-AC52-07NA27344 between the U.S. Department of Energy and LawrenceLivermore National Security, LLC, for the operation of LawrenceLivermore National Security. The Government may have certain rights tothe invention.

FIELD

The present disclosure relates to nanolipoprotein particles (NLPs) and,in particular, to nanolipoprotein particles comprising telodendrimersand Chlamydia major outer membrane protein (MOMP) as well as relatedcompositions, methods and systems.

BACKGROUND

Chlamydia is a prevalent sexually transmitted infection that affectsover 100 million people worldwide. Although most individuals infectedwith Chlamydia trachomatis are initially asymptomatic, symptoms canarise if left undiagnosed. Long-term infection can result indebilitating side effects such as pelvic inflammatory disease,infertility, and blindness. Chlamydia infection, therefore, constitutesa significant public health threat and underscores the need for avaccine.

Chlamydia strains express a major outer membrane protein (MOMP) that isshown to be an effective vaccine antigen. However, in view of poorsolubility, low yield and protein misfolding of the Chlamydia MOMPprotein production of a functional recombinant MOMP protein for vaccinedevelopment has been challenging.

SUMMARY

Provided herein are nanolipoprotein particles comprising Chlamydia majorouter membrane protein (MOMP), and related compositions, methods andsystems which in several embodiments, allow production of soluble andfunctional MOMP antigen and can be used as a vehicle to deliver MOMP ina vaccine.

According to a first aspect, a telodendrimer-nanolipoprotein particle(t-NLP) is described. The t-NLP particle comprises one or more membraneforming lipids, one or more telodendrimers, a scaffold protein and aChlamydia major outer membrane protein (MOMP) or a fragment thereof, theMOMP or the fragment thereof comprising a MOMP hydrophobic region. Inthe telodendrimer-nanolipoprotein particle the one or more membraneforming lipids are arranged in a discoidal membrane lipid bilayerstabilized by the scaffold protein and the one or more telodendrimers,with the membrane lipid bilayer attaching the MOMP or the fragmentthereof through interaction of the MOP hydrophobic region with themembrane lipid bilayer.

According to a second aspect, a method to provide atelodendrimer-nanolipoprotein particle presenting a Chlamydia majorouter membrane proteins (MOMP) and/or a fragment thereof is described,the MOMP and/or the fragment thereof comprising a MOMP hydrophobicregion. The method comprises providing one or more membrane forminglipids, one or more telodendrimers, a polynucleotide coding for the MOMPand/or the fragment thereof and a polynucleotide coding for a scaffoldprotein. The method further comprises mixing the one or more membraneforming lipids and the one or more telodendrimers to provide alipid-telodendrimer mixture, and mixing lipid-telodendrimer mixture withthe polynucleotides and with an in vitro cell free translation system toprovide a single reaction mixture.

The method further comprises translating the polynucleotides within thesingle reaction mixture via the in vitro cell free translation system,the mixing and translating performed to allow self-assembly of thescaffold protein, the one or more membrane forming lipids and the one ormore telodendrimers into a nanolipoprotein particle. In the method, thenanolipoprotein particle comprises the MOMP within a discoidal membranelipid bilayer formed by the one or more membrane forming lipids andstabilized by the scaffold protein, the membrane lipid bilayer attachingthe MOMP through interaction of the target protein hydrophobic regionwith the membrane lipid bilayer.

According to a third aspect, a method to provide atelodendrimer-nanolipoprotein particle presenting a Chlamydia majorouter membrane proteins (MOMP) and/or a fragment thereof is described,the MOMP and/or the fragment thereof comprising a MOMP hydrophobicregion. The method comprises providing one or more membrane forminglipids, one or more telodendrimers, a polynucleotide coding for the MOMPand/or the fragment thereof and a scaffold protein. The method furthercomprises mixing the one or more membrane forming lipids and the one ormore telodendrimers to provide a lipid-telodendrimer mixture, and mixinglipid-telodendrimer mixture with the polynucleotides, the scaffoldprotein with an in vitro cell free translation system to provide asingle reaction mixture.

The method further comprises translating the polynucleotide within thesingle reaction mixture via the in vitro cell free translation system,the mixing and translating performed to allow self-assembly of thescaffold protein, the one or more membrane forming lipids and the one ormore telodendrimers into a nanolipoprotein particle. In the method, thenanolipoprotein particle comprises the MOMP within a discoidal membranelipid bilayer formed by the one or more membrane forming lipids andstabilized by the scaffold protein, the membrane lipid bilayer attachingthe MOMP through interaction of the target protein hydrophobic regionwith the membrane lipid bilayer.

According to a fourth aspect, a system to provide a t-NLP comprisingChlamydia major outer membrane proteins (MOMP) is described. The systemcomprises one or more membrane forming lipids, one or moretelodendrimers, a polynucleotide coding for Chlamydia major outermembrane proteins (MOMP), a polynucleotide coding for a scaffold proteinand/or a scaffold protein for simultaneous combined or sequential use inmethods to provide a t-NLP presenting a MOMP herein described.

According to a fifth aspect, a composition comprising one or moreMOMP-t-NLPs of the present disclosure together with a suitable vehicle,is described. In some embodiments, the composition can further compriseone or more adjuvants. In some embodiments, the vehicle is apharmaceutically acceptable vehicle and the composition is apharmaceutical composition.

According to a sixth aspect, a method and system of immunizing anindividual against Chlamydia is described. The method comprisesadministering to the individual an effective amount a MOMP-t-NLP hereindescribed for a time and under conditions to allow contact of theMOMP-t-NLP with the immune system of the individual. The systemcomprises one or more MOMP t-NLPs herein described together with one ormore adjuvant or adjuvant-NLPs herein described.

According to a seventh aspect, a method and system for treating orpreventing a Chlamydia infection or conditions associated thereto in anindividual, is described, the method comprises administering to theindividual a MOMP-t-NLP herein described in an effective amount toelicit an immunitary response to the MOMP-t-NLPs in the individual. Thesystem comprises one or more MOMP t-NLPs herein described together withone or more adjuvant or adjuvant-NLPs herein described.

Telodendrimer nanolipoproteins and related compositions, methods andsystems, in several embodiments herein described allow, in severalembodiments, production of a soluble recombinant MOMP antigen in afunctional multimeric conformation.

Telodendrimer nanolipoproteins and related compositions, methods andsystems, in several embodiments herein described allow, in severalembodiments, to rapidly produce a high yield recombinant soluble mMOMPexhibiting functional multimer formation.

Telodendrimer nanolipoproteins and related compositions, methods andsystems, in several embodiments herein described allow, in severalembodiments, production of MOMP in particles that can also compriseimmunogenic adjuvants and that can be used in the production of vaccineand/or in methods for generating an immunogenic response in individuals.

Telodendrimer nanolipoproteins and related compositions, methods andsystems, in several embodiments herein described allow, in severalembodiments, immunization against Chlamydia characterized by strongantibody titers.

Telodendrimer nanolipoproteins and related compositions, methods andsystems, in several embodiments herein described can be used, as a modelthat can be applied to other antigens with low solubility (from 0-50% ofthe total amount of antigens in the reaction mixture) or requiring theuse of detergents to first prepare the membrane protein additional tothe scaffold protein for assembly. For example, telodendrimernanolipoproteins and related compositions, methods and systems, inseveral embodiments herein described can be used, as a model for betabarrel forming membrane proteins that form multimeric complexes and tendto form inclusion bodies when over-expressed.

The MOMP-t-NLPs and related compositions, methods and systems hereindescribed can be used in connection with various applications whereinpresentation of functional MOMPs in an ordered structure is desired. Forexample, the MOMP-t-nanolipoprotein particles herein described andrelated compositions methods and systems can be used in antigendetection, generation of functional pores, receptors and membraneenzymes for use as therapeutics as well as immune modulators vaccinedevelopment and use, and/or to contain cell-targeting moieties.Additional exemplary applications include uses of nanolipoproteinparticles in several fields including basic biology research, appliedbiology, bio-engineering, molecular biology, medical research, medicaldiagnostics, structural biology, therapeutics, vaccine development andin additional fields identifiable by a skilled person upon reading ofthe present disclosure.

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, and advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description andexample sections, serve to explain the principles and implementations ofthe disclosure. Exemplary embodiments of the present disclosure willbecome more fully understood from the detailed description and theaccompanying drawings, wherein:

FIG. 1A is a grayscale version of a figure from Feher et al. 2013 [1]which shows a schematic representation of the secondary structure and“predicted topology of the C. trachomatis MOMP serovar C monomer.” (seeFeher et al. 2013 FIG. 4) (SEQ ID NO: 66) wherein the variable domainsare shown by domains with residues in gray circles in the MOMP loops,while transmembrane domains are shown by amino acids within squares.

FIG. 1B is a gray scale version of a figure from Feher et al. [1] whichshows a schematic representation of a “MOMP model surface, mapping ofVD. Positions attaching the VDs to the barrel mapped (dark) onto themolecular surface of the MOMP trimer model” (see Feher et al. 2013 FIG.5A legend) wherein the variable domains are designated by the V1-V4which are known or expected to be important for immunogenicity

FIG. 1C is a gray scale version of a figure from Feher et al. 2013 [1]which shows “MOMP loops 1 and 4 potential inter-monomer stabilizingcontacts. Two of the three monomer b-barrels (light gray on the rightand dark gray on the left ribbon representation) illustrate theproximity of Loops 1 and 4. Their tryptophan and cysteine residues(space-filling atom representation) on neighboring trimer subunits areshown (C29 not modeled). Residues W252 and W273 are at the exteriormembrane interface in the putative aromatic girdle” (see Feher et al.2013 FIG. 5B legend).

FIG. 1D is a figure from Tu et al. 2014 [2]showing “an homology analysisof MOMP multiepitope Grey shading indicates identical residues, andunshaded indicates non-identical residues) (see Tu et al. 2014, FIG. 1legend) (SEQ ID NOs: 67 to SEQ ID NO: 70)

FIG. 2 shows a schematic of an exemplary method to prepare mMOMP-tNLPaccording to an embodiment herein described. FIG. 2 panels (210-250)mMOMP DNA, Δ49ΔpoA1 DNA, and DMPC lipids/telodendrimer were mixed in acell free reaction chamber. FIG. 2 panel (240) Protein translation andthe self-assembly of mMOMP-tNLPs in a cell free lysate. FIG. 2 panel(250) shows a schematic representation of the assembled mMOMP-tNLPproduct. As shown in the schematic, the cell-free expression of mMOMPfrom a DNA construct in presence of Δ49ApoA1 scaffold protein, lipidsand telodendrimers results in the formation of a complex of solublemMOMP in a functional porin structure within a tNLP.

FIGS. 3A-3B shows codon optimized and species matched DNA sequences ofFIG. 3A shows a comparison of wild type vs. codon optimized mousenucleic acid sequence for the encoded Δ49ApoA1 gene (SEQ ID NO: 61, SEQID NO: 62). FIG. 3B shows a comparison between wild type Chlamydiamuridarum MOMP vs. codon optimized MOMP nucleic acid sequence (SEQ IDNO: 64 SEQ ID NO: 65), the asterisk symbol marks where the nativesequence and the optimized sequence are the same. The wild type mousenucleic acid sequence for the encoded Δ49ApoA1 gene is also shown inFIG. 13. The codon optimized mouse nucleic acid sequence for the encodedΔ49ApoA1 gene is also shown in FIG. 14. The wild type Chlamydiamuridarum MOMP nucleic acid sequence is also shown in FIG. 16. The codonoptimized Chlamydia muridarum MOMP nucleic acid sequence is also shownin FIG. 17.

FIGS. 4A-4B shows images of SDS-PAGE gels with separated bands of bodipy(boron dipyrromethene)-labeled mMOMP visualized using a fluorescentimager. The gels show an example of soluble mMOMP production in presenceof tNLPs, exemplified by bodipy-labeled mMOMP expressed in a cell-freesystem. Soluble protein was obtained by centrifuging at 14,100 rcf for10 minutes and supernatant was collected. The total (T) and soluble (S)portions of the cell-free mixture were resolved by SDS-PAGE then imagedwith fluorescent imager. FIG. 4A shows total and soluble portions ofmMOMP produced in a cell-free expression system in the presence of DMPC.FIG. 4B shows total and soluble portions of mMOMP produced in acell-free expression system, co-expressed with Δ49ApoA1 in the presenceof DMPC and telodendrimer PEG^(5k)-CA₈. The presence of the mMOMPprotein band in the ‘S’ lane of FIG. 4B indicates that the addition oftelodendrimers to the cell-free expression system results in solublemMOMP protein, with the solubility of mMOMP increasing from 10% to 75%upon insertion into tNLP.

FIGS. 5A-5C shows examples of expression and purification of mMOMP-tNLP.In particular, FIG. 5A shows a SYPRO Ruby Protein Gel stain of 4-12%SDS-PAGE of nickel affinity chromatography-purified mMOMP-tNLP. Totalunpurified lysate (Total), flow through (FT), Washes (W) 1 and 6, andElutions (E) 1 through 6. All samples were boiled for 5 minutes in thepresence of 50 mM DTT. mMOMP is at 40 kDa and the Δ49ApoA1 is at 22 kDa.M: molecular weight marker. The SDS-PAGE gel densitometry analysis ofFIG. 5A indicates that affinity purified mMOMP-tNLP is 95% pure, andyields 1.5 mg of purified mMOMP-tNLP per 1 mL of cell-free reaction.Distinct bands of mMOMP and Δ49ApoA1 indicate that the two proteins areco-purifying as a complex. FIG. 5B shows a diagram reporting SEC tracesof mMOMP-tNLP particles from four nickel affinity column elutionfractions (E1, E2, E3 and E4), where the different nickel affinitycolumn fractions show the same SEC elution time, indicating they containthe same sized mMOMP-tNLPs. The elutions in panel A and b are the sameelution fractions. X axis represents absorption at 280 nm, whereas Yaxis is the time of elution. The SEC analysis of FIG. 5B confirms thateach mMOMP-tNLP fraction eluted at the appropriate time (retention time(t_(r))˜7 min) without un-incorporated protein or free lipid peaks(tr˜15 min and 4 min, respectively), indicating that the complex was ahomogenous mixture of mMOMP-tNLPs. FIG. 5C shows an image of a dot blotof SEC-purified mMOMP-tNLP samples in duplicate, probed with mAb40 andmAbHIS antibodies. The dot blots of SEC fractions of FIG. 5C showapolipoprotein and mMOMP co-localized within the SEC fractions.

FIGS. 6A-6C show examples of size distribution and cryoEM imaging ofmMOMP-tNLPs. In particular, FIG. 6A shows the size distribution of emptytNLPs as measured by Dynamic Light Scattering (DLS). FIG. 6B shows thesize distribution of mMOMP-tNLPs measured by DLS. The graphs of FIGS. 6Aand 6B show empty tNLPs approximately 10 nm in diameter and mMOMP-tNLPparticle with an almost 4-fold increase in diameter to about 40 nm. FIG.6C shows images of cryoEM of mMOMP-tNLP and tNLP, demonstrating that themMOMP particles are disc like in shape and that there are sizedifferences between tNLP and mMOMP-tNLP particles. Detailed images ofthe discs show the distribution of mMOMP protein multimers, shown asmultiple dark regions of enhanced density within the mMOMP-tNLPparticle.

FIGS. 7A-7B shows images of mMOMP-tNLPs analyzed by SDS-PAGE and Westernblot. In particular, FIG. 7A shows an image of a SYPRO Ruby Protein Gelstain of 4-12% SDS-PAGE of affinity purified mMOMP-tNLP and tNLP alone.mMOMP monomer shows a band at ˜40 kDa and the Δ49ApoA1 shows a band at22 kDa. The mass ratio of soluble mMOMP to apolipoprotein is ˜2:1. Lane1 shows a molecular weight marker (M). In lane 2, the protein bandpattern indicates that the application of heat and reducing agent havebroken down higher order mMOMP structure. In contrast, in lane 3, thepresence of higher order bands indicates mMOMP multimer conformation isretained in absence of the application of heat and reducing agent. FIG.7B shows an image of a Western blot of mMOMP-tNLP and tNLP alone. Shownis a transfer membrane probed with mAb40 (1:1000 dilution). Asindicated, samples were incubated at room temperature or were boiled for5 minutes in the presence of 50 mM DTT. Similar to the results shown inFIG. 7A, the Western blot analysis of FIG. 7B also indicates theformation of higher order oligomer structures of mMOMP in mMOMP-tNLPS,in absence of heat and denaturant.

FIG. 8 shows exemplary results of dot blot analysis of mMOMP-tNLP andtNLP assemblies treated with heat and reducing agent. The mMOMP-tNLP andtNLP assemblies were blotted in triplicate and probed with mAb40 (linearepitope antibody) and mAbHIS (penta-His antibody). Where indicated,samples were boiled for 5 minutes in the presence of 50 mM DTT. In theillustration of FIG. 8, a same intensity of signal is observed forantibodies specific for mMOMP with and without heat and reducing agent,indicating that heat and DTT do not affect the mAb40 binding to mMOMP,and that oligomers of mMOMP are broken down to monomers upon heat andDTT.

FIGS. 9A-9B shows exemplary results of conductance analysis ofmMOMP-tNLPs. In particular, FIG. 9A shows conductance traces recorded at50 mV applied voltage in physiological conditions after tNLP alone (i)and mMOMP-tNLPs (ii-iv) were added to the measurement chamber. Currentincreases seen after mMOMP-tNLP addition indicate pore formation.Representative traces show 1× (ii) and 3× (iii) mMOMP-tNLPincorporations. Trace (iv) shows several incorporations events occurringin quick succession. The increases shown in the traces shown in FIG. 9Aindicate the formation of bilayer pore formation by mMOMP proteins,indicating functional mMOMP insertion. FIG. 9B shows a histogram of 184conductance events. Dashed line indicates best fit to a sum of Gaussianpeaks for 1× and 3× incorporation events, indicating that the mMOMPchannel of FIG. 9B have a tendency to oligomerize within the membraneand form trimers (corresponding to three pores).

FIGS. 10A-10B shows exemplary results of in vivo testing of mMOMP-tNLPs.In particular, FIG. 10A shows results of ELISA analysis, revealing thatmice administered with mMOMP-CpG-tNLPs displayed strong antibody titerscompared to mice administered with tNLPs, CpG-tNLPs, or PBS. Sera frommice administered with mMOMP-CpG-tNLPs, CpG-tNLPs, or PBS were loaded onan ELISA plate pre-coated with mMOMP-tNLPs (black dots) or empty tNLPs(grey dots) and antibody titers were measured. FIG. 10B shows images offour western blots, wherein mMOMP protein was loaded onto each laneequally and mouse sera from mice administered with mMOMP-CpG-tNLPs,CpG-tNLPs, Chlamydia EB, or PBS were then incubated with the blotovernight. Lane 1 is blotted with mouse sera immunized withmMOMP-CpG-tNLP and shows significant mMOMP antibody binding. Lane 2 isblotted with mouse sera immunized with CpG-tNLP. Lane 3 is blotted withmouse sera immunized with live Chlamydia EB and confirms that ChlamydiaEB induces MOMP antibodies that bind to recombinant mMOMP. The decreasedsignal from this blot is because Chlamydia EB contains many surfaceantigens, not just mMOMP, therefore it induces a large variety ofantibodies. Lane 4 is blotted with mice sera immunized with PBS controlgroup and shows no mMOMP binding. M: molecular weight marker.

FIGS. 11A-11B shows a schematic representation of an exemplarytelodendrimer suitable to be included in MOMP-tNLPs herein described. Inparticular, FIG. 11A shows a schematic representation of exemplaryCys-telodendrimer suitable to be included in MOMP-t-NLPs hereindescribed. FIG. 11B shows a schematic representation of exemplaryHis-telodendrimer suitable to be included in MOMP-t-NLPs hereindescribed.

FIGS. 12A-12C shows sequences of scaffold protein and MOMP protein thatcan be used to provide MOMP-NLPs herein described. FIG. 12A shows codonoptimized nucleotide (SEQ ID NO: 55) and amino acid sequence (SEQ ID NO:56) for LLNL mouse Δ49ApoA1. FIG. 12B shows codon optimized nucleotide(SEQ ID NO: 57) and amino acid sequence (SEQ ID NO: 58) for LLNL MouseApoE4, 22 k. FIG. 12C shows codon optimized nucleotide (SEQ ID NO: 59)and amino acid sequence (SEQ ID NO: 60) for LLNL MoPn MOMP based on (NP296436) WP_010232357 and codon optimized.

FIG. 13 shows a wild type mouse nucleic acid sequence (SEQ ID NO: 61)for the encoded Δ49ApoA1 gene.

FIG. 14 shows LLNL codon optimized mouse nucleic acid sequence (SEQ IDNO: 62) for the encoded Δ49ApoA1 gene.

FIG. 15 shows a LLNL codon optimized BALBC mouse nucleic acid sequence(SEQ ID NO: 63) for the encoded Δ49ApoA1 gene.

FIG. 16 shows a wild type Chlamydia muridarum MOMP nucleic acid sequence(SEQ ID NO:64).

FIG. 17 shows LLNL codon optimized Chlamydia muridarum MOMP nucleic acidsequence (SEQ ID NO: 65).

FIGS. 18A-18C shows exemplary results of in vivo testing of MOMP-NLPsformulated with CpG or CpG and FSL₁. FIG. 18A shows antibody tiers fromimmunized mice revealed from ELISA analysis. FIG. 18B shows weight lossof the immunized mice following intranasal challenge with C. muridarum(Cm) as a measure of protection. FIG. 18C plots the number of Cm IFUrecovered from mice vaccinated with different MOMP:NLPs formulations.Each dot represents a mouse. The horizontal line corresponds to themedian. * p<0.05.

FIG. 19 illustrates an exemplary schematic of a Chlamydia vaccinepipeline.

FIG. 20A shows results from the SDS-PAGE analysis following cell-freesynthesis of MOMP-NLPs using varying ratios of fluorescent labeled Apoto MOMP proteins at 1:1, 1:5, 1:10, 1:25, 1:50, and 1:100.

FIG. 20B shows scanning electron microscopic images (SEM) of (A) emptyNLP disc, (B) MOMP-NLP disc with 1-2 monomers of MOMP inserted, (C)MOMP-NLP disc with 1-2 trimers of MOMP inserted, and (D) MOMP-NLP discwith >3 trimers of MOMP inserted.

FIG. 21A shows SDS-PAGE images of codon-optimized fluorescent-labeledPmps (PmpC, PmpE, PmpF, PmpG, and PmpH) having an adhesion domain(annotated as “+”) and truncated versions that lack the adhesion domain(annotated as “−”).

FIG. 21B shows SDS-PAGE images of cell-free expressed andnickel-purified PmpH and ApoA₁ Δ49A₁.

FIGS. 22A-22C demonstrates in an embodiment the cell-free production ofMOMP co-translated with ApoA₁ Δ49A₁. FIG. 22A shows SDS-PAGE images ofcell-free expressed and purified MOMP and ApoA₁ Δ49A₁. FIG. 22B showsexemplary results of dot blot analysis MOMP-NLP and NLP assembliestreated with heat and reducing agent. The MOMP-NLP and NLP assemblieswere blotted in triplicate and probed with mAb40, mAbHIS and mAb18b.Addition of heat and DTT results in a decrease in signal as detected bymAb18b, a conformational trimer recognition antibody, indicating a lossin trimer formation. mAb40 recognizes both MOMP monomer and trimer as itbinds to a linear epitope. The mAbHIS recognizes the HIS tag on theΔ49A1 protein. FIG. 22C shows conductance traces recorded at 50 mVapplied voltage in physiological conditions after NLP alone and MOMP-NLPwere added to the measurement chamber. Current increases observed afterMOMP-NLP addition indicate the formation of bilayer pore formation byMOMP proteins, indicating functional MOMP insertion.

FIGS. 23A-23E shows results of experiments directed to provide astructural characterization of native MOMP. FIG. 23A shows results ofTEM analysis of native MOMP.

FIG. 23B shows TEM images of trimeric MOMP particles selected using asemi-automated particle selection tool via EMAN 2.1 package. FIG. 23Cshows MOMP trimer images be collected and class averaged. FIG. 23D showsraw projection images of MOMP trimer with distinct structural features(white arrows). FIG. 23E shows previously solved MOMP structures, suchas MOMP from Campylobacter jejuni (5 LDT). Black arrow indicates thefitted edges of preliminary density map generated from class averages.

FIG. 24 shows the percentage of weight loss of the immunized micefollowing C. muridarum (Cm) challenge with two different MOMP-t-NLPformulations and two empty NLP formulations.

DETAILED DESCRIPTION

Provided herein are nanolipoprotein particles comprising telodendrimersand Chlamydia major outer membrane protein (MOMP) and relatedcompositions methods and systems.

The term “nanolipoprotein particle” “nanodisc” “rHDL” or “NLP” as usedherein indicates a supramolecular complex formed by a membrane forminglipid arranged in a lipid bilayer stabilized by a scaffold protein. Themembrane forming lipids and scaffold protein are components of the NLP.In particular, the membrane forming lipid component is part of a totallipid component, (herein also membrane lipid component or lipidcomponent) of the NLP together with additional lipids such asfunctionalized lipids and/or lysolipids, that can further be included inthe NLPs as will be understood by a skilled person upon reading of thepresent disclosure. The scaffold protein component is part of a proteincomponent of the NLP together with additional proteins such as membraneproteins, target proteins and other proteins that can be furtherincluded as components of the NLPs as will be understood by a skilledperson upon reading of the present disclosure. Additional components canbe provided as part of the NLP herein described as will be understood bya skilled person. In particular, the membrane lipid bilayer can attachmembrane proteins or other amphipathic compounds through interaction ofrespective hydrophobic regions with the membrane lipid bilayer. Themembrane lipid bilayer can also attach proteins or other moleculethrough anchor compounds or functionalized lipids as will be understoodby a skilled person upon reading of the disclosure. In a nanolipoproteinparticle, the membrane lipid bilayer can be confined in a discoidalconfiguration by the scaffold protein. Predominately discoidal in shape,nanolipoprotein particles typically have diameters between 5 to 25 nm,share uniform heights between 3 to 6 nm and can be produced in yieldsranging between 30 to 90%.

In particular, in embodiments herein described the nanolipoproteinparticle can be formed by a lipid bilayer confined in a discoidalconfiguration by a scaffold protein. In this configuration, the lipidbilayer confined by the scaffold protein can be 3-6 nanometers inthickness, the nanolipoprotein particle can have an overall diameter of5-25 nanometers, and the scaffold protein on the particle can have athickness of 1-2 nanometers. In some embodiments, an entire NLPstructure can be up to 600 kilodaltons in weight.

The particular membrane forming lipid, scaffold protein, the lipid toprotein ratio, and the assembly parameters determine the size andhomogeneity of nanolipoprotein particles as will be understood by askilled person. In the nanolipoprotein particle the membrane forminglipid are typically arranged in a membrane lipid bilayer confined by thescaffold protein in a discoidal configuration as will be understood by askilled person.

The term “membrane forming lipid” or “amphipathic lipid” as used hereinindicates a lipid possessing both hydrophilic and hydrophobic moietiesthat in an aqueous environment assembles into a lipid bilayer structurethat consists of two opposing layers of amphipathic molecules known aspolar lipids. Each polar lipid has a hydrophilic moiety, i.e. a polargroup such as, a derivatized phosphate or a saccharide group, and ahydrophobic moiety, i.e., a long hydrocarbon chain. Exemplary polarlipids include phospholipids, sphingolipids, glycolipids, ether lipids,sterols, alkylphosphocholines and the like. Amphipathic lipids includebut are not limited to membrane lipids, i.e. amphipathic lipids that areconstituents of a biological membrane, such as phospholipids likedimyristoylphosphatidylcholine (DMPC) or dioleoylphosphoethanolamine(DOPE) or dioleoylphosphatidylcholine (DOPC), ordipalmitoylphosphatidylcholine (DPPC). In a preferred embodiment, thelipid is dimyristoylphosphatidylcholine (DMPC).

The term “scaffold protein” as used herein indicates any amphipathicprotein that is capable of self-assembly with amphipathic lipids in anaqueous environment, organizing the amphipathic lipids into a bilayerdisc, and comprise apolipoproteins, lipophorins, derivatives thereof(such as truncated and tandemly arrayed sequences) and fragments thereof(e.g. peptide fragments and synthetic peptides) which maintains theamphipathic nature and capability of self-assembly, such asapolipoprotein E4 (22Kd fragment), lipophorin III, apolipoprotein A-1and the like. In general, scaffold proteins have an alpha helicalsecondary structure in which a plurality of hydrophobic amino acids forma hydrophobic face and a plurality of hydrophilic amino acids form anopposing hydrophilic face. In some embodiments, rationally designedamphipathic peptides and synthetic apolipoproteins which maintain anamphipathic structure and capability of self-assembly can serve as ascaffold protein of the NLP.

The term “apolipoprotein” as used herein indicates an amphipathicprotein that binds lipids to form lipoproteins. The term “amphipathic”pertains to a molecule containing both hydrophilic and hydrophobicproperties. Exemplary amphipathic molecules comprise molecules havinghydrophobic and hydrophilic regions/portions in its structure. Examplesof biomolecules which are amphipathic include but not limited tophospholipids, cholesterol, glycolipids, fatty acids, bile acids,saponins, and additional lipids identifiable by a skilled person. A“lipoprotein” as used herein indicates a biomolecule assembly thatcontains both proteins and lipids. In particular, in lipoproteins, theprotein component surrounds or solubilizes the lipid molecules enablingparticle formation. Exemplary lipoproteins include the plasmalipoprotein particles classified under high-density (HDL) andlow-density (LDL) lipoproteins, which enable fats and cholesterol to becarried in the blood stream, the transmembrane proteins of themitochondrion and the chloroplast, and bacterial lipoproteins. Inparticular, the lipid components of lipoproteins are insoluble in water,but because of their amphipathic properties, apolipoproteins such ascertain Apolipoproteins A and Apolipoproteins B and other amphipathicprotein molecules can organize the lipids in a bilayer orientation withexposed hydrophilic moieties, creating the lipoprotein particle that isitself water-soluble, and can thus be carried through water-basedcirculation (e.g. blood, lymph in vivo or in vitro). Apolipoproteinsknown to provide the protein components of the lipoproteins can bedivided into six classes and several sub-classes, based on the differentstructures and functions. Exemplary apolipoprotein known to be able toform lipoproteins comprise Apolipoproteins A (apo A-I, apo A-II, apoA-IV, and apo A-V), Apolipoproteins B (apo B48 and apo B100),Apolipoproteins C (apo C-I, apo C-II, apo C-III, and apo C-IV),Apolipoproteins D, Apolipoproteins E, and Apolipoproteins H. Forexample, apolipoproteins B can form low-density lipoprotein particles,and have mostly beta-sheet structure and associate with lipid dropletsirreversibly, while Apolipoprotein A1 comprise alpha helices and canassociate with lipid droplets reversibly forming high-densitylipoprotein particles.

The term “protein” as used herein indicates a polypeptide with aparticular secondary and tertiary structure that can interact withanother molecule and in particular, with other biomolecules includingother proteins, DNA, RNA, lipids, metabolites, hormones, chemokines,and/or small molecules. The term “polypeptide” as used herein indicatesan organic linear, circular, or branched polymer composed of two or moreamino acid monomers and/or analogs thereof. The term “polypeptide”includes amino acid polymers of any length including full-lengthproteins and peptides, as well as analogs and fragments thereof. Apolypeptide of three or more amino acids is also called a proteinoligomer, peptide, or oligopeptide. In particular, the terms “peptide”and “oligopeptide” usually indicate a polypeptide with less than 100amino acid monomers. In particular, in a protein, the polypeptideprovides the primary structure of the protein, wherein the term “primarystructure” of a protein refers to the sequence of amino acids in thepolypeptide chain covalently linked to form the polypeptide polymer. Aprotein “sequence” indicates the order of the amino acids that form theprimary structure. Covalent bonds between amino acids within the primarystructure can include peptide bonds or disulfide bonds, and additionalbonds identifiable by a skilled person. Polypeptides in the sense of thepresent disclosure are usually composed of a linear chain of alpha-aminoacid residues covalently linked by peptide bond or a synthetic covalentlinkage. The two ends of the linear polypeptide chain encompassing theterminal residues and the adjacent segment are referred to as thecarboxyl terminus (C-terminus) and the amino terminus (N-terminus) basedon the nature of the free group on each extremity. Unless otherwiseindicated, counting of residues in a polypeptide is performed from theN-terminal end (NH₂-group), which is the end where the amino group isnot involved in a peptide bond to the C-terminal end (—COOH group) whichis the end where a COOH group is not involved in a peptide bond.Proteins and polypeptides can be identified by x-ray crystallography,direct sequencing, immunoprecipitation, and a variety of other methodsas understood by a person skilled in the art. Proteins can be providedin vitro or in vivo by several methods identifiable by a skilled person.In some instances where the proteins are synthetic proteins in at leasta portion of the polymer two or more amino acid monomers and/or analogsthereof are joined through chemically-mediated condensation of anorganic acid (—COOH) and an amine (—NH₂) to form an amide bond or a“peptide” bond.

As used herein the term “amino acid”, “amino acid monomer”, or “aminoacid residue” refers to organic compounds composed of amine andcarboxylic acid functional groups, along with a side-chain specific toeach amino acid. In particular, alpha- or α-amino acid refers to organiccompounds composed of amine (—NH₂) and carboxylic acid (—COOH), and aside-chain specific to each amino acid connected to an alpha carbon.Different amino acids have different side chains and have distinctivecharacteristics, such as charge, polarity, aromaticity, reductionpotential, hydrophobicity, and pKa. Amino acids can be covalently linkedto form a polymer through peptide bonds by reactions between the aminegroup of a first amino acid and the carboxylic acid group of a secondamino acid. Amino acid in the sense of the disclosure refers to any ofthe twenty naturally occurring amino acids, non-natural amino acids, andincludes both D an L optical isomers.

In embodiments herein described, the NLPs herein described furthercomprise one or more telodendrimers to form telo-nanolipoproteinparticles (telo-NLPs or t-NLPs). Predominately discoidal in shape,MOMP-t-NLPS typically have diameters of less than one micron in diameterand in particular can have a diameter from 5 nm to 100 nm in diameter,and in particular from 25 nm to 50 nm. The t-NLPs herein describedtypically have uniform heights between 3 to 6 nm and can be produced inyields ranging between 80 to 90%.

In particular, in embodiments herein described the MOMP-t-NLPs can beformed by a lipid bilayer confined in a discoidal configuration by ascaffold protein and a telodendrimer. In this configuration, the lipidbilayer confined by the scaffold protein can be 3-6 nanometers inthickness, the nanolipoprotein particle can have an overall diameterbetween 5 nm to 100 nm in diameter and in particular a diameter of 25-50nanometers, and the scaffold protein on the particle can have athickness of 1-2 nanometers. In some embodiments, an entire NLPstructure can be up to 600 kilodaltons in molecular weight.

The term “telodendrimer” refers to a dendrimer containing a hydrophiliccovalently attaching a tail group T which comprises a hydrophilicpolymer having a weight averaged molecular weight from 1 to 100 kDa. Theterm “attach” or “attached” as used herein, refers to connecting oruniting by a bond, link, force or tie in order to keep two or morecomponents together, which encompasses either direct or indirectattachment where, for example, a first molecule is directly bound to asecond molecule or material, or one or more intermediate molecules aredisposed between the first molecule and the second molecule or material

The term “dendrimers” used herein refer to repetitively branchedmolecules having three basis architectural components namely (i) a focalpoint or group on a dendrimer core, (ii) repetitive plurality ofbranched monomer units covalently linked to the dendrimer core and (iii)a plurality of end groups each covalently linked to a terminal monomerof the plurality of branched monomer units. In particular, a “dendrimercore” is a chemical moiety presenting a backbone and at least two anchoratoms, each anchor atom defining a bonding position to a head attachmentatom of a branched monomer units.

In some embodiments, the dendrimer core can be formed by a branchedmonomer unit, for example, a lysine unit.

The term “monomer unit” or “monomer” in the sense of the disclosure is achemical structure presenting one head attachment atom and at least onetail attachment atoms. The head attachment atom defines a bondingposition to an anchor atom of a dendrimer core or a tail attachment atomof another monomer unit. The tail attachment atom defines a bondingposition to a head attachment atom of another branch cell unit or to aterminal functional group with the attachment possibly performeddirectly or indirectly.

A “branched monomer unit”, or “branched monomer” is a monomer unithaving at least two tail attachment atoms as also indicated. Ageneration of branched monomer unit within a dendrimer defines a shellof the dendrimer as will be understood by a skilled person (see“Dendrimers and other Dendritic polymers” by Jean M. J Frechet andDonald A. Tomalia 2001 herein incorporated by reference in itsentirety). The branched monomer unit of a generation typically define aninterior space inside the dendrimer herein also indicated as interior ofshell as will be understood by a skilled person. An “end group” of adendrimer, is a functional group or a chemical moiety presented on theoutermost part of the dendrimer attached to an end of branched monomerunit. The branched monomer unit attaching the end groups typicallyprovide the outer shell or periphery of the dendrimer.

In the dendrimer core, the backbone of the dendrimer core can be anystable chemical moiety having the capability to present anchoringpositions for the attachment of branched monomer units and a focal pointfor attachment to a linker moiety L, a spacer moiety A or a tail groupT.

In particular, the core backbone structure can be one of aromatic,heteroaromatic rings, aliphatic, or heteroaliphatic rings or chains. Insome embodiments, the backbone of the dendrimer core can be one singleatom, including C, N, O, S, Si, or P.

In a dendrimer as described herein, the branched monomer unit are linkedtogether to form arms (or “dendrons”) extending from the focal point andterminating at the end groups. The focal point of the dendritic polymercan be attached to other segments of the telodendrimers, and the endgroups may be further functionalized with additional chemical moieties.

In embodiments, herein described, the dendritic polymer can be anysuitable dendritic polymer. The dendritic polymer can be made ofbranched monomer units including amino acids or other bifunctional XY2type monomers, where X and Y are two different functional groups capableof reacting together such that the resulting polymer chain has a branchpoint where an X-Y covalent bond is formed. For example, in the case oflysine, when X is a carboxylic acid and Y is an amino group, an amidebond can be form between X and Y. In some embodiments, each branchedmonomer unit X can be a diamino carboxylic acid, a dihydroxy carboxylicacid and a hydroxylamino carboxylic acid.

In some embodiments, each diamino carboxylic acid can be 2,3-diaminopropanoic acid, 2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid(omithine), 2,6-diaminohexanoic acid (lysine), (2-Aminoethyl)-cysteine,3-amino-2-aminomethyl propanoic acid, 3-amino-2-aminomethyl-2-methylpropanoic acid, 4-amino-2-(2-aminoethyl)butyric acid or5-amino-2-(3-aminopropyl)pentanoic acid. In some embodiments, eachdihydroxy carboxylic acid can be glyceric acid, 2,4-dihydroxybutyricacid, 2,2-Bis(hydroxymethyl)propionic acid,2,2-Bis(hydroxymethyl)butyric acid, serine or threonine.

In some embodiments, each hydroxyl amino carboxylic acid can be serineor homoserine. In some embodiments, the diamino carboxylic acid is anamino acid. In some embodiments, each branched monomer unit X is lysine.

The dendritic polymer of the telodendrimer can be any suitablegeneration of dendrimer, including generation 1, 2, 3, 4, 5, or more,where each “generation” of dendrimer refers to the number of branchpoints encountered between the focal point and the end group followingone branch of the dendrimer. The dendritic polymer of the telodendrimercan also include partial-generations such as 1.5, 2.5, 3.5, 4.5, 5.5,etc., where a branch point of the dendrimer has only a single branch.The various architectures of the dendritic polymer can provide anysuitable number of end groups, including, but not limited to, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31 or 32 end groups.

The telodendrimer backbone can vary, depending on the number of branchesand the number and chemical nature of the end groups and R groups, whichwill modulate solution conformation, rheological properties, and othercharacteristics. The telodendrimers can have any suitable number n ofend groups and any suitable number of R groups. In some embodiments, ncan be 2-70, or 2-50, or 2-30, or 2-10. In some embodiment, n is 2-20.

The R groups installed at the telodendrimer periphery can be anysuitable chemical moiety, including, for example, hydrophilic groups,hydrophobic groups, or amphiphilic compounds. Examples of hydrophobicgroups include, but are not limited to, long-chain alkanes and fattyacids, fluorocarbons, silicones, certain steroids such as cholesterol,and many polymers including, for example, polystyrene and polyisoprene.Examples of hydrophilic groups include, but are not limited to,alcohols, short-chain carboxylic acids, amines, sulfonates, phosphates,sugars, and certain polymers such as PEG. Examples of amphiphiliccompounds include, but are not limited to, molecules that have onehydrophilic face and one hydrophobic face.

Amphiphilic compounds that can be used in the preparation of MOMP-t-NLPsherein described comprise cholic acid and cholic acid analogs andderivatives. “Cholic acid” refers to(R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoicacid. Cholic acid derivatives and analogs comprise allocholic acid,pythocholic acid, avicholic acid, deoxycholic acid, and chenodeoxycholicacid. Cholic acid derivatives can be designed to modulate the propertiesof the nanocarriers resulting from telodendrimer assembly, such asmicelle stability and membrane activity. For example, the cholic acidderivatives can have hydrophilic faces that are modified with one ormore glycerol groups, aminopropanediol groups, or other groups.

In some embodiments, each R of the telodendrimer of formula (I) can becholic acid,(3α,5(3,70α,12α)-7,12-dihydroxy-3-(2,3-dihydroxy-1-propoxy)-cholic acid,(3α,5β,70α,12α)-7-hydroxy-3,12-di(2,3-dihydroxy-1-propoxy)-cholic acid,(3α,5β,7α,12α)-7,12-dihydroxy-3-(3-amino-2-hydroxy-1-propoxy)-cholicacid, cholesterol formate (CF), doxorubicin, or rhein. In someembodiments, each amphiphilic compound is cholic acid (CA). In someembodiments, each amphiphilic compound is cholesterol formate (CF).

In some embodiments, the tail group T can be a moiety of formula (XI)

wherein i and j can be independently selected from 2-3000, preferably22-2300, and more preferably 22-230; andwherein the polymer of Formula (XI) can be attached by way of any one ofthe two terminal hydroxyl groups to an end group of the dendrimer.

In some embodiments, i and j together can be independently selected from2-3000, preferably 22-2300, and more preferably 22-230.

In some embodiments, the tail group can be polyethylene glycol, PEG,(k=0 in formula (XI)), polypropylene glycol (j=0 in Formula XI) or apolyethylene-b-polypropylene glycol (j>0, k>0) in Formula (XI).

In some embodiments herein described, the telodendrimers hereindescribed are block copolymers having a linear poly(ethylene glycol)(PEG) moiety and a dendritic hydrophobic segment or a dendriticamphiphilic moiety. Telodendrimers can also have additional functionalgroups such as cholic acid groups and hydrophobic groups (e.g.hydrophobic moieties with drug properties) covalently bound to thedendritic segment.

As used herein, the term “hydrophobic group” refers to a chemical moietythat is Water-insoluble or repelled by water. Examples of hydrophobicgroups include, but are not limited to, C1-C4 short-chain alkanyls,C5-C22 long-chain alkanyls, C1-C4 short-chain alkenyls, C5-C22long-chain alkenyls, C1-C4 short-chain alkynyls, C5-C22 long-chainalkenyls and fatty acids, fluorocarbons, silicones, certain steroidssuch as cholesterol, and many polymers including, for example,polystyrene and polyisoprene or their derivatives.

As used herein, the term “hydrophilic group” refers to a chemical moietythat is water-soluble or attracted to water. Examples of hydrophilicgroups include, but are not limited to, alcohols, short-chain carboxylicacids, quaternary amines, sulfonates, phosphates, sugars, and certainpolymers such as poly(ethylene glycol) (PEG).

In some embodiments, the PEG as used herein can have 2 to 3000 ethyleneglycol units, —(CH₂CH₂O)—, preferably 22-2300 ethylene glycol units, andmore preferably 22-230 ethylene glycol units.

It is also to be understood that, unless otherwise specified herein, amolecular weight of a polymer herein refers to a weight averagemolecular weight. In the instant disclosure molecular weight of apolymer, e.g. PEG can be indicated as a superscript together with theindication of the polymer (e.g. a PEG of 2000 DA can also be indicatedas PEG^(2k))

As used herein, the term “amphiphilic compound” or “amphiphilic moiety”refers to a compound or moiety having both hydrophobic portions andhydrophilic portions. For example, the amphiphilic compounds hereindescribed can have one hydrophilic face of the compound and onehydrophobic face of the compound.

In some embodiments, in telodendrimers of the disclosure the tail groupT is attached to the dendrimer through a spacer A and/or a linker L.

As used herein the term “spacer A” indicates a spacer moiety formed byone or more monomers configured to be directly covalently connected toone or more tail groups T and to one linker moiety L.

As used herein, the term “linker” or “linker moiety” refers to achemical moiety formed by one or more monomers configured to be directlycovalently bonded to a spacer A and a focal point of a dendrimer. Thetypes of bonds used to link the linker L to the focal point of thedendrimer D and the spacer A include, but are not limited to, amides,amines, esters, carbamates, ureas, thioethers, thiocarbamates,thiocarbonates and thioureas and additional bonds as will be understoodby a skilled person.

In particular, in some embodiments, the telodendrimer of the presentdisclosure can have general formula (I):

(T)_(m)-(A)_(p)-L-D-(R)_(n)  (I)

whereinD is a dendrimerT is a tail group;A is a spacer moiety configured to be directly covalently connected toeach T and to a linker moiety L, and comprises a polymer of 1 to mnumber of spacer A monomers, wherein the spacer A monomer comprises asubstituted or unsubstituted linear C1-C15 alkyl; branched C3-C15 alkyl;cyclic C3-C15 alkyl; linear, cyclic, or branched C2-C15 alkenyl; linear,cyclic, or branched C2-C15 alkynyl; C6-C20 substituted or unsubstitutedaryl; and C6-C20 substituted or unsubstituted heteroaryl.m is 0-20 and p is 0-1, andwherein m is 0 or 1 when p is 0; or m is 2-20 when p is 1;

In some embodiments, L can be a polymer of 1 to m number ofindependently selected spacer A monomers, wherein the spacer A monomercomprises a substituted or unsubstituted linear C1-C15 alkyl; branchedC3-C15 alkyl; cyclic C3-C15 alkyl; linear, cyclic, or branched C2-C15alkenyl; linear, cyclic, or branched C2-C15 alkynyl; C6-C20 substitutedor unsubstituted aryl; and C6-C20 substituted or unsubstitutedheteroaryl, wherein each branch of the dendrimer is adapted to presentan end group R by a covalent bond;

In some of those embodiments, each end group R is independently ahydrophobic group, a hydrophilic group, an amphiphilic group, H, or afunctional group such as halo, hydroxyl, sulfhydryl, C₁-C₂₄ alkoxy,C₂-C₂₄ alkenyloxy, C₂-C₂₄ alkynyloxy, C₅-C₂₄ aryloxy, C₆-C₂₄ aralkyloxy,C₆-C₂₄ alkaryloxy, acyl (including for example C₂-C₂₄ alkylcarbonyl(—CO-alkyl) and C₆-C₂₄ arylcarbonyl (—CO-aryl)), acyloxy (—O-acyl,including for example C₂-C₂₄ alkylcarbonyloxy (—O—CO-alkyl) and C₆-C₂₄arylcarbonyloxy (—O—CO-aryl)), C₂-C₂₄ alkoxycarbonyl (—(CO)—O-alkyl),C₆-C₂₄ aryloxycarbonyl (—(CO)—O-aryl), C₂-C₂₄ alkylcarbonato(—O—(CO)—O-alkyl), C₆-C₂₄ arylcarbonato (—O—(CO)—O-aryl), carboxy(—COOH), carboxylato (—COO—), carbamoyl (—(CO)—NH₂), mono-(C₁-C₂₄alkyl)-substituted carbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)), di-(C₁-C₂₄alkyl)-substituted carbamoyl (—(CO)—N(C₁-C₂₄ alkyl)₂), mono-(C₅-C₂₄aryl)-substituted carbamoyl (—(CO)—NH-aryl), di-(C₅-C₂₄aryl)-substituted carbamoyl (—(CO)—N(C₅-C₂₄ aryl)₂), di-N—(C₁-C₂₄alkyl),N—(C₅-C₂₄ aryl)-substituted carbamoyl, thiocarbamoyl (—(CS)—NH₂),mono-(C₁-C₂₄ alkyl)-substituted thiocarbamoyl (—(CO)—NH(C₁-C₂₄ alkyl)),di-(C₁-C₂₄ alkyl)-substituted thiocarbamoyl (—(CO)—N(C₁-C₂₄ alkyl)₂),mono-(C₅-C₂₄ aryl)-substituted thiocarbamoyl (—(CO)—NH-aryl), di-(C₅-C₂₄aryl)-substituted thiocarbamoyl (—(CO)—N(C₅-C₂₄ aryl)₂), di-N—(C₁-C₂₄alkyl),N—(C₅-C₂₄ aryl)-substituted thiocarbamoyl, carbamido(—NH—(CO)—NH₂), cyano(—C≡N), cyanato (—O—C≡N), thiocyanato (—S—C≡N),formyl (—(CO)—H), thioformyl (—(CS)—H), amino (—NH₂), mono-(C₁-C₂₄alkyl)-substituted amino, di-(C₁-C₂₄ alkyl)-substituted amino,mono-(C₅-C₂₄ aryl)-substituted amino, di-(C₅-C₂₄ aryl)-substitutedamino, C₂-C₂₄ alkylamido (—NH—(CO)-alkyl), C₆-C₂₄ arylamido(—NH—(CO)-aryl), imino (—CR═NH where R=hydrogen, C₁-C₂₄ alkyl, C₅-C₂₄aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl, etc.), C₂-C₂₀ alkylimino(—CR═N(alkyl), where R=hydrogen, C₁-C₂₄ alkyl, C₅-C₂₄ aryl, C₆-C₂₄alkaryl, C₆-C₂₄ aralkyl, etc.), arylimino (—CR═N(aryl), whereR=hydrogen, C₁-C₂₀ alkyl, C₅-C₂₄ aryl, C₆-C₂₄ alkaryl, C₆-C₂₄ aralkyl,etc.), nitro (—NO₂), nitroso (—NO), sulfo (—SO₂—OH), sulfonato(—SO₂—O—), C₁-C₂₄ alkylsulfanyl (—S-alkyl; also termed “alkylthio”),C₅-C₂₄ arylsulfanyl (—S-aryl; also termed “arylthio”), C₁-C₂₄alkylsulfinyl (—(SO)-alkyl), C₅-C₂₄ arylsulfinyl (—(SO)-aryl), C₁-C₂₄alkylsulfonyl (—SO₂-alkyl), C₅-C₂₄ arylsulfonyl (—SO₂-aryl), boryl(—BH₂), borono (—B(OH)₂), boronato (—B(OR)₂ where R is alkyl or otherhydrocarbyl), phosphono (—P(O)(OH)₂), phosphonato (—P(O)(O—)₂),phosphinato (—P(O)(O—)), phospho (—PO₂), phosphino (—PH2), silyl (—SiR₃wherein R is hydrogen or hydrocarbyl), and silyloxy (—O-silyl); and thehydrocarbyl moieties C₁-C₂₄ alkyl (preferably C₁-C₁₂ alkyl, morepreferably C₁-C₆ alkyl), C₂-C₂₄ alkenyl (preferably C₂-C₁₂ alkenyl, morepreferably C₂-C₆ alkenyl), C₂-C₂₄ alkynyl (preferably C₂-C₁₂ alkynyl,more preferably C₂-C₆ alkynyl), C₅-C₂₄ aryl (preferably C₅-C₁₄ aryl),C₆-C₂₄ alkaryl (preferably C₆-C₁₆ alkaryl), and C₆-C₂₄ aralkyl(preferably C₆-C₁₆ aralkyl).

In some embodiments, the tail group T is polyethyleneglycol (PEG)polymers, each of the m number PEG polymer independently having a weightaverage molecular weight of 1-100 kDa.

In some embodiments, a telodendrimer can have the at least one tailgroup T having polyethyleneglycol (PEG) polymer moiety, a dendriticpolymer moiety D, and at least one end group R which includes but is notlimited to a hydrophobic group, a hydrophilic group, an amphiphiliccompound or a drug on the dendrimer periphery or branch, wherein thedendritic polymer moiety D has a single focal group and n number ofbranches.

In some embodiments, a telodendrimer can comprise one or more of thefollowing monomers in combination within a dendrimer, spacer moiety Aand/or linker moiety be XY2-type monomers, where X and Y are twodifferent functional groups capable of reacting together such that theresulting polymer chain has a branch point where an X-Y bond is formed.Exemplary monomers include a diamino carboxylic acid, a dihydroxycarboxylic acid and a hydroxyl amino carboxylic acid. Examples ofdiamino carboxylic acid groups include 2,3-diamino propanoic acid,2,4-diaminobutanoic acid, 2,5-diaminopentanoic acid (ornithine),2,6-diaminohexanoic acid (lysine), (2-Aminoethyl)-cysteine,3-amino-2-aminomethyl propanoic acid, 3-amino-2-aminomethyl-2-methylpropanoic acid, 4-amino-2-(2-aminoethyl)butyric acid and5-amino-2-(3-aminopropyl)pentanoic acid. Examples of dihydroxycarboxylic acid groups include glyceric acid, 2,4-dihydroxybutyric acid,and 2,2-bis(hydroxymethyl)propionic acid. Examples of hydroxyl aminocarboxylic acids include serine and homoserine. One of skill in the artwill appreciate other monomer units useful in the current disclosure.

In addition, the aforementioned functional groups may, if a particulargroup permits, be further substituted with one or more additionalfunctional groups or with one or more hydrocarbyl moieties such as thosespecifically enumerated above. Analogously, the above-mentionedhydrocarbyl moieties may be further substituted with one or morefunctional groups or additional hydrocarbyl moieties such as thosespecifically enumerated.

In some embodiments, in formula (I) subscript n is an integer from 2 to128, wherein subscript n is equal to the number of end group R; whereineach end group R is covalently linked to the dendritic polymer D, andwherein at least half the number n of R groups are each independently ahydrophobic group, a hydrophilic group, an amphiphilic group or a drug.

In formula (I) subscript p can be 0 or 1, wherein when p is 0, m can be0 or 1; when p is 1, m can be 2 to 20 wherein each of the m number ofPEG is directly covalently linked to A and each of the m number of PEGsis independently selected from a molecular weight of 1 to 100 kDa, orpreferably a molecular weight of 1 kDa (PEG1000) to a molecular weightof 10 kDa (PEG 10,000).

In some embodiments, spacer moiety A can be a monomer or an oligomerpresenting to at least two tail groups. As used herein, the terms“monomer” and “monomer unit” for spacer moiety A refers to repeatingunits that make up the spacer moiety A herein described. The monomersmay be XY2-type monomers, where X and Y are two different functionalgroups capable of reacting together such that the resulting polymerchain has a branch point where an X-Y bond is formed.

For purpose of making spacer moiety A, one of the two Y's of a XY2-typemonomer can be orthogonally protected, for example by way of Fmoc(Fluorenylmethyloxycarbonyl), Boc (t-butyloxycarbonyl), or DDE((4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl) when B is an aminogroup and A is a carboxylic acid.

Therefore, each of the XY2 in spacer moiety A is capable of having acovalent bond with a tail group T.

Exemplary monomers for spacer moiety A include a diamino carboxylicacid, a dihydroxy carboxylic acid and a hydroxylamino carboxylic acid.Examples of diamino carboxylic acid groups herein described comprise 2,3diamino propanoic acid, 2,4-diaminobutanoic acid, 2,5-di aminopentanoicacid (omithine), 2,6-diaminohexanoic acid (lysine),(2-Aminoethyl)-cysteine, 3-amino-2-aminomethyl propanoic acid,3-amino-2-aminomethyl-2-methyl propanoic acid,4-amino-2-(2-aminoethyl)butyric acid and5-amino-2-(3-aminopropyl)pentanoic acid. Examples of dihydroxycarboxylic acid groups of telodendrimers of the present disclosurecomprise glyceric acid, 2,4-dihydroxy butyric acid, and2,2-bis(hydroxymethyl)propionic acid. Examples of hydroxylaminocarboxylic acids include, but are not limited to, serine and homoserineas well as additional monomeric units as will be understood by a skilledperson.

In some embodiments, spacer moiety A comprises an oligomer of lysinerepresented by (K)_(m″) wherein oligomer of lysine has a peptidebackbone based on an alpha amino group of lysine, wherein K is lysineand m″ is 1-20 and wherein m″ is an integer between m−1 to 20. In someembodiment, m″ is m−1.

In some embodiment, at least one of the dendrimer, spacer moiety Aand/or linker moiety L can independently comprise at least one monomerselected from XY2-type monomers, where A and B are two differentfunctional groups capable of reacting together such that the resultingpolymer chain has a branch point where an X-Y bond is formed. Exemplarymonomers include a diamino carboxylic acid, a dihydroxy carboxylic acidand a hydroxylamino carboxylic acid. Examples of diamino carboxylic acidgroups herein described comprise 2,3 diamino propanoic acid,2,4-diaminobutanoic acid, 2,5-di aminopentanoic acid (omithine),2,6-diaminohexanoic acid (lysine), (2-Aminoethyl)-cysteine,3-amino-2-aminomethyl propanoic acid, 3-amino-2-aminomethyl-2-methylpropanoic acid, 4-amino-2-(2-aminoethyl)butyric acid and5-amino-2-(3-aminopropyl)pentanoic acid. Examples of dihydroxycarboxylic acid groups of telodendrimers of the present disclosurecomprise glyceric acid, 2,4-dihydroxy butyric acid, and2,2-bis(hydroxymethyl)propionic acid. Examples of hydroxylaminocarboxylic acids include, but are not limited to, serine and homoserineas well as additional monomeric units as will be understood by a skilledperson.

In some embodiments a dendrimer can comprise branched polymerscontaining a focal point, a plurality of branched monomer units, and aplurality of end groups in which the focal point of the dendriticpolymer is a functional group on the branched monomer that is of equalspacing from all the end groups can be attached to another segment ofthe telodendrimer, including linker L, spacer A or tail group T. The endgroups may be further functionalized with additional chemical moieties.

In embodiments wherein the telodendrimer has formula (I), the focalpoint of a telodendrimer or a telodendrimer segment can be any suitablefunctional group that form a covalent bond between the dendrimer and atail group T, spacer moiety A, a linker moiety L.

In some embodiments, the functional group for the focal point can be anucleophilic group including, but not limited to, an alcohol, an amine,a thiol, or a hydrazine. The focal point functional group can also be anelectrophile such as an aldehyde, a carboxylic acid, or a carboxylicacid derivative including for example an acid chloride or anN-hydroxysuccinimidyl ester.

The telodendrimer of formula (I) can have a single type of R group onthe periphery, or any combination of R groups in any suitable ratio. Ingeneral, at least half the number n of R groups are other than an endgroup. For example, at least half the number n of R groups can be ahydrophobic group, a hydrophilic group, an amphiphilic compound, a drug,or any combination thereof. In some embodiments, half the number n of Rgroups are amphiphilic compounds.

In some embodiments, all the R groups are an amphiphilic group such ascholic acid or cholesterol formate. In other embodiments, some of the Rgroups are an end group of the dendrimer. In some other embodiments, atleast two different R groups are present, such as two differentamphiphilic groups, or an amphiphilic group and a drug, or anamphiphilic group and a dendritic polymer end group, or two differentdrugs, or a drug and a dendritic end group.

In some embodiments, telodendrimers of t-NLPs of Formula (I), D can belysine, L can be a bond, R can be cholic acid or cholate, m can be 1,and/or n can be 2, 4 or 8. In some embodiments, R can be formed by adetergent moiety, a lipid and/or an amino acid such as HIS, GLU.

In some embodiments, the telodendrimer of the present disclosurecomprise a compound of formulas (II)-(III):

PEG-D-(R)_(n)  (II)

PEG-L-D-(R)_(n)  (III)

(PEG)_(m′)-A-L-D-(R)_(n)  (IV)

wherein D, L, R and n are as defined for formula (I) and subscript m′ offormula (IV) is 2-20.

In some embodiments, the PEG in telodendrimer of any one of formula (I)to (IV) can be a PEG having a molecular weight from 1 kDA (PEG1000) to10 kDA (PEG 10,000).

In some embodiments, MOMP-t-NLPs herein described can comprisetelodendrimers such as PEG^(2K)-D-CA₄, PEG^(5K)-D-CA₄, PEG^(10K)-D-CA₄,PEG^(2K)-D-CA₈, PEG^(5K)-D-CA₈, PEG^(10K)-D-CA₈, PEG^(2K)-D-CF₄,PEG^(5K)-D-CF₄, PEG^(10K)-D-CF₄, PEG^(2K)-D-CF₈, PEG^(5K)-D-CF₈, orPEG^(10K)-D-CF₈, wherein each dendritic polymer D is a poly(lysine)dendritic polymer wherein each end group is hydroxy. In one embodiment,the telodendrimer can be PEG^(5K)-D-CF₈. Additional modifications forthe telodendrimer can include attachment of lipidic and detergentmoieties such as Telo-His and Telo-Cys.

In some embodiments, MOMP-t-NLPs herein described can comprisetelodendrimers such as PEG^(2K)-D-CA₄, PEG^(5K)-D-CA₄, PEG^(10K)-D-CA₄,PEG^(2K)-D-CA₈, PEG^(5K)-D-CA₈, PEG^(10K)-D-CA₈, PEG^(2K)-D-CF₄,PEG^(5K)-D-CF₄, PEG^(10K)-D-CF₄, PEG^(2K)-D-CF₈, PEG^(5K)-D-CF₈, orPEG^(10K)-D-CF₈, wherein each dendritic polymer D is a poly(lysine)dendritic polymer wherein each end group is hydroxy. In one embodiment,the telodendrimer can be PEG^(5K)-D-CF₈. Additional modifications forthe telodendrimer can include attachment of lipidic and detergentmoieties such as Telo-His and Telo-Cys.

A schematic representation of an exemplary telodendrimer comprising atelo-cys is shown in FIG. 11A. A schematic representation of anexemplary telodenrimer comprising telo-His is shown in FIG. 11B. In someembodiments, in a Telo-Cys according to the schematic representation ofFIG. 11A, or a in a Telo-His according to the schematic of FIG. 10B, thecholic acid is covalently linked to a cysteine amine or a lysine byamide bond; cysteine and lysine are or lysine and lysine are covalentlyconnected by an amide bond and a core lysine monomer is covalentlyattached to a tail group of PEG 5000. In some embodiments,telodendrimers herein described can comprise a combination of cysteineand histidine as will be understood by a skilled person.

In some embodiments, an Ebes linker,(N-(Fmoc-8-amino-3,6-dioxa-octyl)succinamic acid), is present betweenthe tail group PEG 5000 and the core lysine monomer by amide bond and anester bond.

In particular, in preferred embodiments, MOMP-t-NLPs comprising one ormore of PEG^(5K)-D-CA₄, PEG^(5K)-D-CA₈, PEG^(5K)-D-CF₄, andPEG^(5K)-D-CF₈, provided an improved formulation of MOMP proteins withina tNLP compared to other telodendrimers herein described. Thetelodendrimers useful in the preparation of t-NLPs herein described canbe prepared by a variety of methods, such as those described in PCTPublication No. WO 2010/039496 herein incorporated by reference in itsentirety.

In embodiments herein described the nanolipoprotein particles furthercomprise a Chlamydia major outer membrane protein (MOMP).

The term “Chlamydia” as used herein indicates a genus of pathogenicbacteria of the phylum Chlamydiae that are obligate intracellularbacteria as well as the bacteria belonging to said genus. Chlamydiabacteria are ovoid in shape and stain Gram-negative. Chlamydia bacteriaare characterized by a developmental cycle involving an infectiouselementary body (EB) and the vegetative reticulate body (RB). Inparticular, the EB remains within a phagosome after Chlamydia attachesand promotes entry into a target host cell. The EB differentiates intothe RB which then redifferentiate into EB after several rounds ofreplication. The EB is small, dense, rigid, metabolically inert, andresistant to the hostile extracellular environment while the RB islarge, low-density, less rigid, metabolically active but noninfectious(Moulder, J. W., Hatch, T. P., Kuo, C. C., Schachter, J., and Storz, J.1984. Order II: Chlamydiales. In Bergey's manual of systematicbacteriology, Vol. 1 (eds. N. R. Krieg and J. G. Holt), pp. 729-739.Williams & Wilkins, Baltimore, Md.). Chlamydia comprise Chlamydiaspecies Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydiapsittaci (human pathogens), Chlamydia suis (affects only swine),Chlamydia pecorum (affects cows/swine/koala) and Chlamydia pneumonia(affects koala) and Chlamydia muridarum (affects only mice and hamsters)

The term “MOMP” as used herein indicates the major outer membraneprotein of a bacterium of the genus Chlamydia capable of folding into abeta barrel structure that can associate with other MOMP proteins. MOMPcan be encoded by the gene ompA of bacteria of the Chlamydia genus.Typically, a MOMP beta barrel structure consists of 18 transmembraneregions. In general, MOMP has a molecular mass of ˜40 kDa and can makeup 60% of total outer membrane protein. Chlamydial MOMP is detectableboth in the EB and in the RB of Chlamydia with techniques such asmonoclonal antibodies (MAbs) and surface radioiodination as well asadditional techniques identifiable by a skilled person. MOMPs compriseproteins with low solubility (from 0% to 50% of the total amount of MOMPprotein in the mixture). In particular, MOMP can have a solubility scorelower or equal to 20% and in some instances a solubility of 10% orlower.

MOMP has been identified to be a porin even if MOMP has been associatedwith other functions such as a potential chlamydial cytoadhesin as wellas a structural protein. Porins are a family of membrane channelscommonly found in the outer membranes of Gram-negative bacteria, wherethey serve as diffusion pathways for nutrients, waste products, andantibiotics and can also be receptors for bacteriophages. Porins have astructural topology comprised of antiparallel β-strands spanning theouter membrane, a water-filled inner channel, tight β-turns extendinginto the periplasmic region and flexible loops reaching beyond theextracellular surface [1]. The MOMP of Chlamydia genus contains foursymmetrically spaced variable domains (VDs 1 to 4). The variable domainregions are predicted to be outside the trans-membrane β-strands.Detailed structural description of MOMP of Chlamydia can be found inFeher et al. 2014 [1].

MOMP in the sense of the disclosure encompasses a protein from aChlamydia bacterium capable of oligomerization, formation ofhomo-trimers and functional porins, and capable of forming antigens thatcan elicit an immune response. In some embodiments, MOMP comprised intNLPs described herein primarily forms homo-trimers [3].

MOMP in the sense of the disclosure encompass MOMP proteins of variousbacteria within the Chlamydia genus as well as species-specific variantsof MOMP, such as a MoPn MOMP protein (mMOMP), a type of MOMP expressedin the mouse-specific bacterium Chlamydia muridarum,

Sequence information from various strains and species within theChlamydia genus can be accessed via the National Center forBiotechnology Information website as will be understood by a personskilled in the art. For example, sequence information for the Chlamydiamuridarum MOMP gene (ompA) can be accessed via the National Center forBiotechnology Information website at the addresshttps://www.ncbi.nlm.nih.gov/nuccore/U60196. Sequence information forthe Chlamydia trachomatis MOMP gene (ompA) can be accessed via theNational Center for Biotechnology Information website at the addresshttps://www.ncbi.nlm.nih.gov/gene/884473. Exemplary MOMP gene andprotein sequences are listed in Table 1.

TABLE1 Exemplary MOMPgene and protein sequences Origin SequencesSEQ ID NO Chlamydia muridarumATGAAAAAACTCTTGAAATCGGTATTAGCATTTGCCGTTTTGGGTTCTGCT 1 MOMP geneTCCTCCTTGCATGCTCTGCCTGTGGGGAATCCTGCTGAACCAAGCCTTATG (ompA)ATTGACGGGATTCTTTGGGAAGGTTTCGGTGGAGATCCTTGCGATCCTTGCACAACTTGGTGTGATGCCATCAGCCTACGTCTCGGCTACTATGGGGACTTCGTTTTTGATCGTGTTTTGAAAACAGACGTGAACAAACAGTTCGAAATGGGAGCAGCTCCTACAGGAGATGCAGACCTTACTACAGCACCTACTCCTGCATCAAGAGAGAATCCCGCTTATGGCAAGCATATGCAAGATGCAGAAATGTTCACTAATGCTGCGTACATGGCTTTAAACATTTGGGACCGTTTCGATGTATTTTGTACATTGGGAGCAACTAGCGGATATCTTAAAGGTAATTCTGCCGCCTTTAACTTAGTTGGTCTGTTTGGAAGAGATGAAACTGCAGTTGCAGCTGACGACATACCTAACGTCAGCTTGTCTCAAGCTGTTGTCGAACTCTACACAGACACAGCTTTCGCTTGGAGCGTCGGTGCTAGAGCAGCTTTATGGGAGTGCGGATGTGCAACTTTAGGAGCTTCCTTCCAATATGCTCAATCTAAGCCAAAAGTAGAGGAATTAAACGTTCTCTGTAATGCGGCAGAATTCACTATTAACAAGCCTAAAGGATACGTTGGACAAGAGTTTCCTCTTAACATTAAAGCTGGAACAGTTAGCGCTACAGATACTAAAGATGCTTCCATCGATTACCATGAGTGGCAAGCAAGCTTGGCTTTGTCTTACAGACTGAATATGTTCACTCCTTACATTGGAGTTAAGTGGTCTAGAGCAAGCTTTGATGCCGACACTATCCGCATTGCGCAGCCTAAGCTTGAGACCTCTATCTTAAAAATGACCACTTGGAACCCAACGATCTCTGGATCTGGTATAGACGTTGATACAAAAATCACGGATACATTACAAATTGTTTCCTTGCAGCTCAACAAGATGAAATCCAGAAAATCTTGCGGTCTTGCAATTGGAACAACAATTGTAGATGCTGATAAATATGCAGTTACTGTTGAGACACGCTTGATCGATGAAAGAGCAGCTCACGTAAATGCTCAGTTCCGTTTCTAA ChlamydiaMKKLLKSVLAFAVLGSASSLHALPVGNPAEPSLMEDGILWEGFGGDPCDPC 2 muridarum MOMPTTWCDAISLRLGYYGDFVFDRVLKTDVNKQPEMGAAPTGDADLTTAPTPAS protein (GenBank:RENPAYGKHMQDAEMFTNAAYMALNIWDRFDVFCTLGATSGYLKGNSAAFN AAB07068.1)LVGLFGRDETAVAADDIPNVSLSQAVVELYTDTAFAWSVGARAALWECGCATLGASFQYAQSKPKVEELNVLCNAAEFTINKPKGYVGQEFPLNIKAGTVSATDTKDASIDYHEWQASLALSYRLNMFTPYIGVKWSRASFDADTIRIAQPKLETSILKMTTWNPTISGSGEDVDTKITDTLQIVSLQLNKMKSRKSCGLAIGTTIVDADKYAVTVETRLEDERAAHVNAQFRF ChlamydiaATGAAAAAACTCTTGAAATCGGTATTAGTATTTGCCGCTTTGAGTTCTGCT 3 trachomatis strainTCCTCCTTGCAAGCTCTGCCTGTGGGGAATCCTGCTGAACCAAGCCTTATG A/Har-1 major outerATCGACGGAATTCTGTGGGAAGGTTTCGGCGGAGATCCTTGCGATCCTTGC membrane proteinACCACTTGGTGTGACGCTATCAGCATGCGTATGGGTTACTATGGTGACTTT (ompA) geneGTTTTCGACCGTGTTTTGAAAACAGATGTGAATAAAGAATTTCAGTAGGGA (GenBank:GCGGCGCCTACTACCAGCGATGTAGCAGGCTTAGAAAAGGATCCAGTAGCA DQ064279.1)AATGTTGCTCGCCCAAATCCCGCTTATGGCAAACACATGCAAGATGCTGAAATGTTTACGAACGCTGCTTACATGGCATTAAATATCTGGGATCGTTTTGATGTATTTTGTACATTGGGAGCAACTACCGGTTATTTAAAAGGAAACTCCGCTTCCTTCAACTTAGTTGGATTATTCGGAACAAAAACACAATCTTCTGGCTTTGATACAGCGAATATTGTTCCTAACACTGCTTTGAATCAAGCTGTGGTTGAGCTTTATACAGACACTACCTTTGCTTGGAGCGTAGGTGCTCGTGCAGCTCTCTGGGAATGTGGGTGTGCAACGTTAGGAGCTTCTTTCCAATATGCTCAATCTAAACCTAAAGTAGAAGAGTTGAATGTTCTTTGTAATGCATCCGAATTTACTATTAATAAGCCGAAAGGATATGTTGGGGCGGAATTTCCACTTGATATTACCGCAGGAACAGAAGCTGCGACAGGGACTAAGGATGCCTCTATTGACTACCATGAGTGGCAAGCAAGTTTAGCCCTTTCTTACAGATTAAATATGTTCACTCCTTACATTGGAGTTAAATGGTCTAGAGTAAGTTTTGATGCCGACACGATCCGTATCGCTCAGCCTAAATTGGCTAAACCAGTCTTGGATACCACTACTCTAAACCCGACCATCGCTGGTAAAGGAACTGTGGTCTCTTCCGCAGAAAACGAACTGGCTGATACAATGCAAATCGTTTCCTTGCAGTTGAACAAGATGAAATCTAGAAAATCTTGCGGTATTGCAGTAGGAACAACTGTTGTAGATGCAGATAAATACGCAGTTACAATTGAGACTCGCTTGATCGATGAGAGAGCAGCTCACGTAAAT GCACAATTCCGCTTCTAAChlamydia MKKLLKSVLVFAALSSASSLQALPVGNPAEPSLMEDGILWEGFGGDPCDPC 4trachomatis strain TTWCDAISMRMGYYGDFVFDRVLKTDVNKEFQMGAAPTTSDVAGLEKDPVAA/Har-1 major outer NVARPNPAYGKHMQDAEMFTNAAYMALNIWDRFDVFCTLGATTGYLKGNSAmembrane protein SFNLVGLFGTKTQSSGFDTANIVPNTALNQAVVELYTDTTFAWSVGARAALsequence (ompA) WECGCATLGASFQYAQSKPKVEELNVLCNASEFTINKPKGYVGAEFPLDITAGTEAATGTKDASIDYHEWQASLALSYRLNMFTPYIGVKWSRVSFDADTIRIAQPKLAKPVLDTTTLNPTIAGKGTVVSSAENELADTMQIVSLQLNKMKSRKSCGIAVGTTVVDADKYAVTIETRLIDERAAHVNAQFRF ChlamydiaATGAAAAAACTCTTGAAATCGGTATTAGTATTTGCCGCTTTGAGTTCTGCT 5 trachomatis strainTCCTCCTTGCAAGCTCTGCCTGTGGGGAATCCTGCTGAACCAAGCCTTATG B/Tunis-864 majorATCGACGGAATTCTGTGGGAAGGTTTCGGCGGAGATCCTTGCGATCCTTGC outer membraneACCACTTGGTGTGACGCTATCAGCATGCGTATGGGTTACTATGGTGACTTT protein (ompA)GTTTTCGACCGTGTTTTGAAAACAGATGTGAATAAAGAATTCCAAATGGGT gene, complete cdsGCCAAGCCTACAGCTACTACAGGCAATGCTACAGCTCCATCCACTCTTACA (Genbank:GCAAGAGAGAATCCTGCTTACGGCCGACATATGCAGGATGCTGAGATGTTT DQ064280.1)ACAAATGCCGCTTGCATGGCATTGAATATTTGGGATCGCTTTGATGTATTCTGTACACTAGGAGCCTCTAGCGGATACCTTAAAGGAAACTCTGCTTCTTTCAATTTAGTGGGGTTATTCGGAAATAATGAGAACCAGACTAAAGTTTCAAATGGTACGTTTGTACCAAATATGAGCTTAGATCAATCTGTTGTTGAGTTGTATACAGATACTGCTTTTGCGTGGAGCGTCGGCGCTCGCGCAGCTTTGTGGGAATGTGGATGTGCAACTTTAGGAGCTTCTTTCCAATATGCTCAATCTAAACCTAAAGTAGAAGAATTAAACGTTCTCTGCAATGCAGCAGAGTTTACTATTAATAAACCTAAAGGGTATGTAGGTAAGGAGTTGCCTCTTGATCTTACAGCAGGAACAGATGCTGCGACAGGAACTAAGGATGCCTCTATTGATTACCATGAATGGCAAGCAAGTTTAGCTCTCTCTTACAGATTGAATATGTTCACTCCTTACATTGGAGTTAAATGGTCTCGAGCAAGCTTTGATGCAGACACGATTCGTATTGCTCAGCCGAAGTCAGCCGAGACTATCTTTGATGTTACCACTCTGAACCCAACTATTGCTGGAGCTGGCGATGTGAAAACTAGCGCAGAGGGTCAGCTCGGAGACACAATGCAAATCGTCTCCTTGCAATTGAACAAGATGAAATCTAGAAAATCTTGCGGTATTGCAGTAGGAACAACTATTGTGGATGCAGACAAATACGCAGTTACAGTTGAGACTCGCTTGATCGATGAGAGAGCTGCTCACGTAAATGCACAA TTCCGCTTCTAAChlamydia MKKLLKSVLVFAALSSASSLQALPVGNPAEPSLMIDGILWEGFGGDPCDPC 6trachomatis strain TTWCDAISMRMGYYGDFVFDRVLKTDVNKEFQMGAKPTATTGNATAPSTLTB/Tunis-864 major ARENPAYGRHMQDAEMFTNAACMALNIWDRFDVFCTLGASSGYLKGNSASFouter membrane NLVGLFGNNENQTKVSNGTFVPNMSLDQSVVELYTDTAFAWSVGARAALWEprotein sequence CGCATLGASFQYAQSKPKVEELNVLCNAAEFTINKPKGYVGKELPLDLTAG(ompA) TDAATGTKDASIDYHEWQASLALSYRLNMFTPYIGVKWSRASFDADTIRIAQPKSAETIFDVTTLNPTIAGAGDVKTSAEGQLGDTMQIVSLQLNKMKSRKSCGIAVGTTIVDADKYAVTVETRLIDERAAHVNAQFRF

In some embodiments, the MOMP-NLPs herein described can include one ormore MOMP fragments alone or in combination with MOMP protein. The term“MOMP fragment” is a portion of a MOMP protein herein describedcomprising a transmembrane region including for example MOMP hydrophobicamino acid configured to interact with a membrane lipid bilayer. In aMOMP fragment, the transmembrane region can be formed by at least onetransmembrane domain each domain comprising 3 to 40 hydrophobic aminoacid residues.

Additional sequences of the ompA gene and MOMP protein or fragmentsthereof are recognizable by persons skilled in the art, comprisingsequences of members of the Chlamydia genus such as Chlamydiatrachomatis, Chlamydia muridarum and Chlamydia suis and in particularthe fifteen known Chlamydia trachomatis serovars and additional strainsknown to those skilled in the art, which can be found in publicdatabases such as NCBI.

In particular, C. trachomatis includes three human biovars: (1) SerovarsAb, B, Ba, or C, which cause trachoma: infection of the eyes, which canlead to blindness, (2) Serovars D-K, which cause urethritis, pelvicinflammatory disease, ectopic pregnancy, neonatal pneumonia, andneonatal conjunctivitis, and (3) Serovars L1, L2, and L3, which causelymphogranuloma venereum.

The term “biovar” as used herein refers to a variant prokaryotic strainthat differs physiologically and/or biochemically from other strains ina particular species. The term “serovar” refers to strains that haveantigenic properties that differ from other strains.

In some embodiments, additional ompA genes and MOMP proteins can beidentified by conducting homology search of gene or protein sequences indatabases such as NCBI and others known to persons skilled in the art.

Homology can be determined using available sequence analysis algorithmprograms including but not limited to CLUSTAL, ALIGN, GAP, BESTFIT,BLAST, FASTA, and TFASTA among others known to a skilled person.Sequences of DNA, mRNA, or protein having at least 50% sequence identityto known ompA DNA and mRNA sequences and MOMP protein sequences can beconsidered homologous. The term “percent identity” refers to aquantitative measurement of the similarity between sequences of apolypeptide or a polynucleotide and, in particular, indicates the amountof characters that match between two different sequences. The similaritybetween sequences is typically measured by a process that comprises thesteps of aligning the two polypeptide or polynucleotide sequences toform aligned sequences, then detecting the number of matched characters,i.e. characters similar or identical between the two aligned sequences,and calculating the total number of matched characters divided by thetotal number of aligned characters in each polypeptide or polynucleotidesequence, including gaps. The similarity result is expressed as apercentage of identity.

Homology can also be determined on the basis of protein structuralsimilarity. Several publicly available online servers can be used todetect protein structure alignment and calculate percent structuralsimilarity, such as FATCAT, SuperPose, iPBA, MAPSCI, and others known toa person skilled in the art. Proteins having at least 50% structuralidentity to known MOMP protein structures or fragments thereof can beconsidered homologous.

MOMP in the sense of the disclosure also includes codon-optimizedsequences of MOMP expressed in a cell-free expression system, hereinexemplified by an E. coli cell-free expression system (see e.g. thesequences illustrated in FIGS. 3A-3B and the sequences illustrated inFIGS. 12C, 16 and 17). Additional MOMP variants comprise MOMPs having asequence that differs in all or in part from a naturally occurring MOMPand that maintain the beta barrel structure with the 18 transmembraneregions separated by loops. In particular, additional MOMP variantsinclude modifications with respect to a natural MOMP protein whichmaintain the barrel structure while changing the level of immunogenicityof the MOMP. In some of these embodiments, the difference in sequencebetween a natural MOMP and the variant can be localized in the extracellular loops.

MOMP in the sense of the present disclosure comprise wild type MOMP andMOMP derivatives such as MOMP including mutations, deletions,truncations and MOMP fusion protein including MOMP fused with otherpeptides.

In some embodiments, the MOMP in the sense of the present disclosure canbe recombinant forms of MOMP in which the MOMP coding sequence has beenmutated to present a unique DNA sequence, which does not alter the aminoacids to enhance transcription and translation of the target protein.Exemplary sequences are shown in the illustration of FIGS. 3A-3B and arealso shown in FIGS. 16 and 17. The exemplary mMOMP gene of FIGS. 3A-3B(also shown in FIG. 17) provide an E. coli codon-optimized version ofMOMP expressed in the mouse-specific Chlamydia muridarum. The exemplaryMoPn gene and protein sequences of FIG. 12C provide an E. colicodon-optimized version of MOMP expressed in the mouse-specificChlamydia muridarum MoPn.

As would be understood by those skilled in the art, the term “codonoptimization” as used herein refers to the introduction of synonymousmutations into codons of a protein-coding gene in order to improveprotein expression in expression systems of a particular organism, suchas E. coli in accordance with the codon usage bias of that organism. Theterm “codon usage bias” refers to differences in the frequency ofoccurrence of synonymous codons in coding DNA. The genetic codes ofdifferent organisms are often biased towards using one of the severalcodons that encode a same amino acid over others—thus using the onecodon with, a greater frequency than expected by chance. Optimizedcodons in microorganisms, such as Escherichia coli or Saccharomycescerevisiae, reflect the composition of their respective genomic tRNApool. The use of optimized codons can help to achieve faster translationrates and high accuracy.

In the field of bioinformatics and computational biology, manystatistical methods have been proposed and used to analyze codon usagebias. Methods such as the ‘frequency of optimal codons’ (Fop), theRelative Codon Adaptation (RCA) or the ‘Codon Adaptation Index’ (CAI)are used to predict gene expression levels, while methods such as the‘effective number of codons’ (Nc) and Shannon entropy from informationtheory are used to measure codon usage evenness. Multivariatestatistical methods, such as correspondence analysis and principalcomponent analysis, are widely used to analyze variations in codon usageamong genes. There are many computer programs to implement thestatistical analyses enumerated above, including CodonW, GCUA, INCA, andothers identifiable by those skilled in the art. Codon optimization hasapplications in designing synthetic genes and DNA vaccines. Severalsoftware packages are available online for codon optimization of genesequences, including those offered by companies such as GenScript, EnCorBiotechnology, Integrated DNA Technologies, ThermoFisher Scientific,among others known those skilled in the art. Those packages can be usedin providing MOMP with codon ensuring optimized expression in variouscell systems as will be understood by a skilled person.

In particular, MOMP in the sense of the disclosure can comprisemonomeric or multimeric MOMP such as dimeric and trimeric MOMP.

In some embodiments, MOMP-t-NLPs herein described comprise multimericMOMP (>2 membrane proteins) embedded in nanoparticles.

In some embodiments, MOMP-t-NLP herein described comprise at least oneMOMP from Chlamydia species Chlamydia trachomatis Chlamydia pneumoniae,and Chlamydia psittaci (human pathogens), Chlamydia suis (affects onlyswine), Chlamydia pecorum (affects cows/swine/koala) and Chlamydiapneumonia (affects koala) and Chlamydia muridarum (affects only mice andhamsters) or a variant thereof

In some embodiments, the membrane forming lipids component of the lipidcomponent lipids such as phospholipids, preferably including at leastone phospholipid, typically soy phosphatidylcholine, eggphosphatidylcholine, soy phosphatidylglycerol, egg phosphatidylglycerol,palmitoyl-oleoyl-phosphatidylcholine distearoylphosphatidylcholine, ordistearoylphosphatidylglycerol. Other useful phospholipids include,e.g., phosphatidylcholine, phosphatidylglycerol, sphingomyelin,phosphatidylserine, phosphatidic acid, phosphatidylethanolamine,lysolecithin, lysophosphatidylethanolamine, phosphatidylinositol,cephalin, cardiolipin, cerebrosides, dicetylphosphate,dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine,dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol,stearoyl-palmitoyl-phosphatidylcholine,di-palmitoyl-phosphatidylethanolamine,distearoyl-phosphatidylethanolamine, di-myrstoyl-phosphatidylserine,di-myrstoyl-phosphatidylserine and dioleyl-phosphatidylcholine.

Additionally exemplary membrane forming lipids that can be comprised invarious combinations together with one or more lysolipids comprise1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-dilauroyl-sn-glycero-3-phosphocholine,1,2-didecanoyl-sn-glycero-3-phosphocholine,1,2-dierucoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine,1,2-dimyristoleoyl-sn-glycero-3-phosphocholine,1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine,1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, eggphosphatidylcholine extracts, soy phosphatidylcholine extracts, heartphosphatidylcholine extracts, brain phosphatidylcholine extracts, liverphosphatidylcholine extracts, 1,2-distearoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dilauroyl-sn-glycero-3-phosphate,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate,1-stearoyl-2-oleoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine,1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine,1,2-dilauroyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, Eggphosphatidylethanolamine extract, soy phosphatidylethanolamine extract,heart phosphatidylethanolamine extract, brain phosphatidylethanolamineextract, 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol),1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol),1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol),1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol),1,2-dilauroyl-sn-glycero-3-phospho-(1′-rac-glycerol),1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol), eggphosphatidylglycerol extract, soy phosphatidylglycerol extract,1,2-distearoyl-sn-glycero-3-phospho-L-serine,1,2-dioleoyl-sn-glycero-3-phospho-L-serine,1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine,1,2-dimyristoyl-sn-glycero-3-phospho-L-serine,1,2-dilauroyl-sn-glycero-3-phospho-L-serine,1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine, soyphosphatidylserine extract, brain phosphatidylserine extract,2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate,cholesterol, ergosterol, sphingolipids, ceramides, sphingomyelin,gangliosides, glycosphingolipids,1,2-dioleoyl-3-trimethylammonium-propane,1,2-di-O-octadecenyl-3-trimethylammonium propane.

In some embodiments, non-phosphorus containing lipids can also be usedas membrane forming lipids in the MOMP-t-NLPs herein described, e.g.stearylamine, docecylamine, acetyl palmitate, and fatty acid amides.Additional membrane forming lipids suitable for use in providing NLPsare well known to persons of ordinary skill in the art and are cited ina variety of well-known sources, e.g., McCutcheon's Detergents andEmulsifiers and McCutcheon's Functional Materials, Allured PublishingCo., Ridgewood, N.J., both of which are incorporated herein byreference.

In some embodiments, the scaffold proteins can contain amino acidadditions, deletions, or substitutions. In other embodiments, thescaffold proteins can be derived from various species and moreparticularly derived from human, mouse, rat, guinea pig, rabbit, cow,horse, pig, dog, koala, and non-human primates.

In some embodiments membrane forming lipids can be comprised within aMOMP-t-NLP stabilized by scaffold proteins such as human derived apoE4,truncated versions of human derived apoE4 (e.g. apoE422k), human derivedapoE3, truncated versions of human derived apoE3 (e.g. apoE322k), humanderived apoE2, truncated versions of human derived apoE2 (e.g.apoE222k), human derived apoA1, truncated versions of human derivedapoA1 (e.g. Δ49ApoA1, MSP1, MSP1T2, MSP1E3D1), mouse derived apoE4,truncated versions of mouse derived apoE4 (e.g. apoE422k), mouse derivedapoE3, truncated versions of mouse derived apoE3 (e.g. apoE322k), mousederived apoE2, truncated versions of mouse derived apoE2 (e.g.apoE222k), mouse derived apoA1, truncated versions of mouse derivedapoA1 (e.g. Δ49ApoA1, MSP1, MSP1T2, MSP1E3D1), rat derived apoE4,truncated versions of rat derived apoE4 (e.g. apoE422k), rat derivedapoE3, truncated versions of rat derived apoE3 (e.g. apoE322k), ratderived apoE2, truncated versions of rat derived apoE2 (e.g. apoE222k),rat derived apoA1, truncated versions of rat derived apoA1 (e.g.Δ49ApoA1, MSP1, MSP1T2, MSP1E3D1), lipophorins (e.g. B. mori, M. sexta),synthetic cyclic peptides that mimic the function of apolipoproteins.Other apolipoproteins, as will be understood for a skilled person, canbe used to form NLP, including but not limited to apoB and apoC.

In some embodiments, the scaffold protein can be codon-optimized inorder to improve protein expression in expression systems of aparticular organism. Exemplary polynucleotide and amino acid sequencesof E. coli codon optimized scaffold protein are shown in FIGS. 12A and12B. Exemplary polynucleotide sequences of E. coli codon optimizedscaffold protein are shown in FIGS. 14 and 15.

In some embodiments, the scaffold protein is formed by amphipathicpeptides and/or synthetic apolipoproteins which are configured tomaintain an amphipathic structure and capability of self-assembly. Inparticular, in those embodiments, the peptides and/or syntheticapolipoprotein are configured and selected to provide the a plurality ofhelical segments each having a primary structure configured to form analpha helix secondary structure, In the alpha helix secondary structureof at least one helical segment, the peptides and/or syntheticapolipoprotein comprise a plurality of hydrophobic amino acids and aplurality of hydrophilic amino acids positioned in the primary structureto provide an amphipathic alpha helix secondary structure, with theplurality of hydrophobic amino acids forming an hydrophobic amino acidcluster and the plurality hydrophilic amino acids forming an hydrophilicamino acid cluster. In some of those embodiments, the scaffold proteinscan be peptides derived from apolipoproteins, and can contain amino acidadditions, deletions, or substitutions. In other embodiments, thesepeptides have no sequence homology to apolipoproteins but can bestructural analogs. In some embodiments, the peptides can be preparedwith L- or D-amino acids. In embodiments where the scaffold proteincomprises one or more peptides the skilled person would be able toidentify the ratios of peptides based on the length and number ofpeptides and apolipoproteins and on a desired dimension of thenanolipoprotein particles upon reading of the present disclosure.Additional description of scaffold proteins can be found inPCT/US2015/051172 published on Mar. 16, 2017 as WO2017/044899incorporated herein by reference in its entirety.

In several embodiments herein described, MOMP-t-NLPs show differentsize, compositions, and homogeneity. Composition of a t-NLP can bedetected by various techniques known in the art, such as highperformance liquid chromatography (HPLC), reverse phase high performanceliquid chromatography (RP-HPLC), mass spectrometry, thin layerchromatography, NMR spectroscopy and elemental analysis could be used todefine the composition of the particles and additional techniquesidentifiable by a skilled person.

Size and compositions of the MOMP-t-NLPs can be characterized by SEC(size exclusion chromatography) traces which are used to separate outmolecules in solution by their size and in some cases their molecularweights as will be understood by a skilled person.

In some embodiments, a MOMP-t-NLP herein described can have a sizeranging between 5 nm to 100 nm in diameter. In some embodiments, aMOMP-t-NLP herein described can have a size ranging between 10 nm to 70nm in diameter. In some embodiments, a MOMP-t-NLP herein described canhave a size ranging between 25 nm to 50 nm in diameter

In embodiments herein described, NLPs comprise scaffold protein and alipid component comprising membrane forming lipids and possibly otherlipids, as well telodendrimers and MOMP in ratios and proportions thatwould be identifiable by a skilled person upon reading of the presentdisclosure.

In general, assembly of telo-NLPs can be accomplished with a wide rangeof ratios of total membrane forming lipids to scaffold proteins aspreviously described. Telodendrimer can be incorporated at a ratio of1:10 to 1:1000 telodendrimer to lipid, with a preferred ratio between1:50 and 1:500, or more preferably between 1:100 and 1:200.

The t-NLPs here described can contain any suitable combination of lipidswith telodendrimers and/or other components. In particular, the one ormore membrane forming lipids mixed to form a t-NLP can be polar and/ornon-polar lipids as will be understood by a skilled person upon readingof the present disclosure. The telodendrimers mixed to form the t-NLPscan comprise PEG with lengths of 1000-10000 kDa. The ratio of lipid totelodendrimer in the t-NLPs, for example, can be from about 1000:1 toabout 10:1 (mol/mol). For example, the ratio can be about 1000:1, 900:1,800:1, 700:1, 600:1, 500:1, 400:1, 300:1, 200:1, 100:1, 99:1, 95:1,90:1, 80:1, 75:1, 70:1, 60:1, 50:1, 40:1, 30:1, 25:1, 20:1, 15:1, 14:1,13:1, 12:1, 11:1 or 10:1 (mol/mol) wherein the term about when referredto ratios indicates the ratios ±5%. In some embodiments, the ratio oflipid to telodendrimer is from about 200:1 to about 100:1 (mol/mol). Insome embodiments, the ratio of lipid to telodendrimer is about 150:1(mol/mol). In some embodiments, the ratio of lipid to telodendrimer isabout 135:1 (W/W). Other molar ratios of lipid to telodendrimer can alsobe useful in t-NLPs herein described as will be apparent to a skilledperson upon reading of the present disclosure. In some embodiments oft-NLPs, the lipid to telodendrimer ratios within the telo-NLPs hereindescribed can be of 1000:1 to 10:1, preferably 50:1 to 500:1

In some embodiments, a MOMP-t-NLP herein described, can have a ratio ofscaffold protein to lipid is 1:30 to 1:100.

In some embodiments, a MOMP-t-NLP herein described can have a ratio ofMOMP to scaffold protein of 50:1 to 1:10 (see, for example, Example 9and FIGS. 20A-B). In some embodiments, the ratio of MOMP to scaffoldprotein can be 20:1 to 1:4, 5:1 to 1:2 or of 3:1 to 1:1.

In some embodiments, a MOMP-t-NLP herein described can have a ratio ofMOMP to NLPs of 1:1 to 50:1. In some embodiments, the ratio of MOMP toNLPs is 1:1 to 3:1 or 6:1, 9:1 and 12:1.

Any measuring technique available in the art can be used to determineproperties of the t-NLPs herein described. For example, techniques suchas size exclusion chromatography (SEC), small angle X-ray scattering(SAXS), dynamic light scattering (DLS), x-ray photoelectron microscopy,powder x-ray diffraction, scanning electron microscopy (SEM),transmission electron microscopy (TEM), cryo-electron microscopy(cryo-EM), and atomic force microscopy (AFM) can be used to determineaverage size and dispersity of the t-NLPs.

In preferred embodiments, a MOMP-t-NLP herein described can have a sizeranging between 5 nm to 100 nm in diameter with a ratio of telodendrimerto lipid is 1:10 to 1:1000, a ratio of scaffold protein to lipid of 1:30to 1:100 and a ratio of MOMP to scaffold protein is 20:1 to 1:4.

More preferably among the most preferred embodiments, a MOMP-t-NLPherein described can have a size ranging between 10 nm to 70 nm indiameter with a ratio of telodendrimer to lipid 1:50 to 1:500, a ratioof scaffold protein to lipid 1:30 to 1:100, and a ratio of MOMP toscaffold protein 5:1 to 1:2.

In most preferred embodiments, a MOMP-t-NLP herein described has a sizeranges between 25 nm to 50 nm in diameter. In the MOMP-t-NLP, the ratioof telodendrimer to lipid is 1:100 to 1:200, the ratio of scaffoldprotein to lipid is 1:30 to 1:100, and the ratio of MOMP to scaffoldprotein is 3:1 to 1:1.

In those embodiments, MOMP-t-NLPs can solubilize a MOMP with asolubility score ≤20% of the total amount of the MOMP protein in themixture.

In particular, MOMP-t-NLP with the above preferred and in particular,most preferred ratios are capable of increasing a MOMP's solubility froma solubility score of 10% to a solubility score greater than 70% whenembedded in the resulting t-NLP-MOMP particle, the percentage calculatedwith respect to the total amount of the MOMP protein in the mixture

In some of these embodiments, the increase in solubility allows MOMPprotein yield to be as high as 2 mg/mL cell-free reaction, and MOMPinsertion rate in the final construct to be as high as 50% or greaterwith respect to the total amount of MOMP in the reaction mixture

In particular, in some embodiments, MOMP-t-NLP with the above preferredand in particular, most preferred ratios can provide an increase of5-50% for the solubility of MOMP assembled in to a tNLP compared to thesolubility of MOMP in a mixture in absence of tNLP. Once the material ispurified, all of the subsequent material is present at 100% solubility.

Additionally, some embodiments of the MOMP-t-NLPs with the above ratioscan allow oligomer MOMP protein to be embedded in a single water solublenanoparticle, as well as the generation of 25 nm to 50 nm sizenanoparticle suitable for in vivo application. Additionally, MOMP-t-NLPswith the above ratios are particularly suitable in compositions, methodsand systems directed to elicit an immunogenic response against MOMP inan individual.

In some embodiments, the MOMP-t-NLPs show a larger than expected size ofapproximately 40 nm then previously identified using other methods.

In some embodiments, MOMP-t-NLPs herein described can further includeadditional lipids such as functionalized amphipathic compounds and/orone or more target proteins that can be added during the assembly of thet-NLP herein described such as polymorphic membrane proteins (PMP) thatmay interact with MOMP.

The term “Polymorphic Membrane Proteins” as described herein indicates agroup of membrane-bound, surface-exposed chlamydial proteins that haverepetitive domains, cell binding domains and beta barrel membrane bounddomain as will be understood by a skilled person. In particular, in someof these embodiments, MOMP-t-NLPs herein described comprise PMPs thatare capable of forming and/or form disulfide-bond-cross-linked proteinswith MOMPs and/or other PMPs. In particular, these PMPS share nohomology with other bacterial proteins but do share common featuresamong Chlamydia spp.

In some embodiments herein described, the MOMP-t-NLPs further comprisefull-length Pmps from a same or different Chlamydial species such asChlamydia muridarum or C. trachomatis. In particular, in someembodiments, the MOMP-t-NLPs can further comprise any combination of pmpproteins PmpA, PmpB, PmpC, PmpD, PmpE, PmpF, PmpG, PmpH, and/or PmpIfrom that C. muridarum and/or C. trachomatis as will be understood by aperson of ordinary skill in the art. In particular the MOMP-t-NLPs cancomprise one or more Pmp proteins in a same or different MOMP-t-NLPswithin a composition comprising one or more MOMP-t-NLPs hereindescribed. In particular in some embodiments, the MOMP-t-NLPs hereindescribed can comprise one or more of Pmp C, E, F, G and H. In someembodiments, the MOMP-t-NLPs herein described can comprise one or moreof Pmp A, B, D and I.

In some embodiments, MOMPs are co-translated with one or more Pmps in acell-free method/system in presence of NLPs components to formMOMP-Pmp-t-NLPs (see Example 10). Vaccination with MOMP-Pmp-t-NLPs canprovide enhanced immunogenic protection against Chlamydia infection.

In some embodiments, a MOMP-Pmp-t-NLP herein described can have a ratioof scaffold protein to lipid is 1:30 to 1:100.

In some embodiments, a MOMP-Pmp-t-NLP herein described can have a ratioof Pmps to scaffold protein of 50:1 to 1:10. In some embodiments, theratio of Pmps to scaffold protein can be 20:1 to 1:4, 5:1 to 1:2 or of3:1 to 1:1.

In some embodiments, a MOMP-Pmp-t-NLP herein described can have a ratioof Pmps to NLPs of 1:1 to 50:1. In some embodiments, the ratio of Pmpsto NLPs is 1:1 to 3:1 or 6:1, 9:1 and 12:1.

In preferred embodiments, a MOMP-Pmp-t-NLP herein described can have asize ranging between 5 nm to 100 nm in diameter with a ratio oftelodendrimer to lipid is 1:10 to 1:1000, a ratio of scaffold protein tolipid of 1:30 to 1:100, a ratio of MOMP to scaffold protein is 20:1 to1:4, and a ratio of Pmps to scaffold protein is 20:1 to 1:4.

More preferably among the most preferred embodiments, a MOMP-Pmp-t-NLPherein described can have a size ranging between 10 nm to 70 nm indiameter with a ratio of telodendrimer to lipid 1:50 to 1:500, a ratioof scaffold protein to lipid 1:30 to 1:100, a ratio of MOMP to scaffoldprotein 5:1 to 1:2, and a ratio of Pmps to scaffold protein 5:1 to 1:2.

In most preferred embodiments, a MOMP-Pmp-t-NLP herein described has asize ranges between 25 nm to 50 nm in diameter. In the MOMP-Pmp-t-NLP,the ratio of telodendrimer to lipid is 1:100 to 1:200, the ratio ofscaffold protein to lipid is 1:30 to 1:100, the ratio of MOMP toscaffold protein is 3:1 to 1:1, and the ratio of Pmps to scaffoldprotein is 3:1 to 1:1.

The term “functionalized amphipathic compounds” in the sense of thedisclosure indicates compounds having a hydrophobic portion and ahydrophilic portion in a configuration where the hydrophobic portionanchor is able to anchor the compound to the lipid bilayer of the NLPand the hydrophilic portion is presented on the NLP bilayer facefollowing NLP assembly. In the functionalized amphipathic compounds inthe sense of the disclosure the hydrophilic portion of typicallyessentially consists of or comprises a hydrophilic functional group.

The term “functional group” as used herein indicates specific groups ofatoms within a molecular structure that are responsible for acharacteristic chemical reaction of that structure. Exemplary functionalgroups include hydrocarbons, groups containing double or triple bonds,groups containing halogen, groups containing oxygen, groups containingnitrogen and groups containing phosphorus and sulfur all identifiable bya skilled person.

The term “present” as used herein with reference to a compound orfunctional group indicates attachment performed to maintain the chemicalreactivity of the compound or functional group as attached. Accordingly,a functional group presented on an amphipathic compound, is able toperform under the appropriate conditions the one or more chemicalreactions that chemically characterize the functional group.

The use of functionalized amphipathic compounds enables attachment ofvarious peptides or other biologics to the surfaces of the lipid of theNLP that allows some desired target features to be obtained, such asstability, affinity for a target molecule, and the like. Non-limitingexamples of functional groups presented on functionalized lipidsinclude: chelated Ni atoms, azide, anhydride, alkynes, thiols, halogens,carboxy, amino, hydroxyl, and phosphate groups, and additional groupsidentifiable by a skilled person upon reading of the present disclosure.

In some embodiments, the functional group on the functionalizedamphipathic compound can be a reactive chemical groups (e.g. azide,chelated nickel, alkyne, and additional reactive chemical groupsidentifiable by a skilled person), a biologically active compound (e.g.DNA, peptide, carbohydrate, and additional biologically active groupidentifiable by a skilled person) or a small molecule (e.g. cellulartargeting compound, adjuvant, drug, and additional small moleculesidentifiable by a skilled person). In some embodiments, thefunctionalized amphipathic compound is a functionalized lipid compound.Functional groups that enhance the lipid solubility are referred to ashydrophobic or lipophilic functional groups. Functional groups that lackthe ability to either ionize or form hydrogen bonds tend to impart ameasure of lipid solubility to a drug molecule. The functional group canbe attached to the lipid polar head through covalent or ionic bonds and“weak bonds” such as dipole-dipole interactions, the London dispersionforce and hydrogen bonding, preferably covalent. Moreover,functionalization of the lipid can involve hydrophobic quantum dotsembedded into the lipid bilayer. The following article is incorporatedby reference in its entirety: R. A. Sperling, and W. J. Parak. “Surfacemodification, functionalization and bioconjugation of colloidalinorganic nanoparticles”. Phil. Trans. R. Soc. A 28 Mar. 2010 vol. 368no. 1915 1333-1383 [4].

In some embodiments, functionalized amphipathic compounds can compriseone or more of1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-((folate)amino)hexanoyl),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(6-azidohexanoyl),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(dodecanyl),1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(hexanoylamine),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(dodecanylamine),1,2-Dipalmitoyl-sn-Glycero-3-Phosphothioethanol,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide],1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide],1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate],1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl),1,2-Dioleoyl-sn-Glycero-3-Phospho(Ethylene Glycol),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[dibenzocyclooctyl(polyethyleneglycol)-2000],1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[succinyl(polyethyleneglycol)-2000],1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)-2000],1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000],1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethyleneglycol)-2000],1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000],1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000],1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[cyanur(polyethyleneglycol)-2000],1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[folate(polyethyleneglycol)-2000], cholesterol modified oligonucleotides,cholesterol-PEG2000-azide, cholesterol-PEG2000-Dibenzocyclooctyl,cholesterol-PEG2000-maleimide, cholesterol-PEG2000-N-hydroxysuccinimideesters, cholesterol-PEG2000-thiol, cholesterol-azide,cholesterol-Dibenzocyclooctyl, cholesterol-maleimide,cholesterol-N-hydroxysuccinimide esters, cholesterol-thiol, C18 modifiedoligonucleotides, C18-PEG2000-azide, C18-PEG2000-Dibenzocyclooctyl,C18-PEG2000-maleimide, C18-PEG2000-N-hydroxysuccinimide esters,C18-PEG2000-thiol, C18-azide, C18-Dibenzocyclooctyl, C18-maleimide,C18-N-hydroxysuccinimide esters, C18-thiol.

In some embodiments, the MOMP-telo-nanolipoprotein particles hereindescribed can further comprise one or more membrane proteins herein alsoindicated as target protein. The term “membrane protein” as used hereinindicates any protein having a structure that is suitable for attachmentto or association with a biological membrane or biomembrane (i.e. anenclosing or separating amphipathic layer that acts as a barrier withinor around a cell). In particular, membrane proteins include proteinsthat contain large regions or structural domains that are hydrophobic(the regions that are embedded in or bound to the membrane); thoseproteins can be difficult to work with in aqueous systems, since whenremoved from their normal lipid bilayer environment those proteins tendto aggregate and become insoluble.

Methods and systems for production of MOMP-t-NLPs are also described. Inthe methods and systems herein described expression of MOMP and thescaffold protein of a MOMP-t-NLP herein described is performed in acell-free method/system in presence of other NLPs components for a timeand under conditions that allow assembly of the NLP.

The membrane forming lipid and the protein components of the MOMP-t-NLPare generally able to self-assemble in a biological (largely aqueous)environment according to the thermodynamics associated with waterexclusion (increasing entropy) during hydrophobic association. In themethods and systems herein provided, the amphipathic lipid and theprotein components of the NLP are allowed to assembly in a cell freeexpression system.

As used herein, the wording “cell free expression”, “cell freetranslation”, “in vitro translation” or “IVT” refer to at least onecompound or reagent that, when combined with a polynucleotide encoding apolypeptide of interest, allows in vitro translation of saidpolypeptide/protein of interest.

The term “polynucleotide” as used herein indicates an organic polymercomposed of two or more monomers including nucleotides, or analogsthereof. The term “nucleotide” refers to any of several compounds thatconsist of a ribose (ribonucleotide) or deoxyribose(deoxyribonucleotides) sugar joined to a purine or pyrimidine base andto a phosphate group, and that are the basic structural units of nucleicacids. The term “nucleotide analog” refers to a nucleotide in which oneor more individual atoms have been replaced with a different atom with adifferent functional group. Accordingly, the term polynucleotideincludes nucleic acids of any length of DNA or RNA analogs and fragmentsthereof. A polynucleotide of three or more nucleotides is also callednucleotidic oligomers or oligonucleotide.

In particular, co-expression of both scaffold protein and MOMP inpresence of phospholipids with or without surfactant/detergent can beperformed in a “one-pot” reaction that generates, in situ, both scaffoldprotein and target membrane protein; NLP self-assembly will ensue usingphospholipids already in the reaction mixture.

In some embodiments, the additives used in the cell free reactionsystems include any substance that improves the solubilization of theprotein of interest and/or of any other protein components that arepresent in the reaction mixtures, any substance that may augment proteinproduction and any substance that improves protein functions. Thoseadditives include but are not limited to cofactors (e.g. retinal, heme)other proteins that facilitate modification (e.g. glycosylases,phosphatases, chaperonins) lipids, redox factors, detergents andprotease inhibitors, and in particular, phospholipids such asdimyristoylphosphatidyl choline (DMPC) and the like, andsurfactants/detergents such as cholate, triton X-100 and the like.Exemplary detergents that can be used for protein solubilization in themethods and systems herein disclosed, includeHeptanoyl-N-methyl-glucamide, Octanoyl-N-methyl-glucamide,Nonanoyl-Nmethyl-glucamide, n-Nonyl-b-D-gluco-pyranoside,N-Octyl-b-D-glucopyranoside, Octyl-b-D-thiogluco-pyranoside,NN-Dimethyldodecylamine-N-oxide and Glycerol. Additional additives thatmight be included in the reaction mixtures include labels and labelingmolecule that can be used to label or tag the target protein and thus toenable the detection of the target protein through detection of arelated labeling signal.

The terms “label” and “labeled molecule” as used herein refer to amolecule capable of detection, including but not limited to radioactiveisotopes, fluorophores, chemiluminescent dyes, chromophores, enzymes,enzymes substrates, enzyme cofactors, enzyme inhibitors, dyes, metalions, nanoparticles, metal sols, ligands (such as biotin, avidin,streptavidin or haptens) and the like. The term “fluorophore” refers toa substance or a portion thereof which is capable of exhibitingfluorescence in a detectable image. As a consequence, the wording“labeling signal” as used herein indicates the signal emitted from thelabel that allows detection of the label, including but not limited toradioactivity, fluorescence, chemoluminescence, production of a compoundin outcome of an enzymatic reaction and the like.

In some embodiments, the polynucleotides encoding MOMP and/or thescaffold protein or other proteins can comprise an engineeredpolynucleotide designed such that the resulting protein can be expressedas a full-length protein. In some embodiments, the polynucleotide is anengineered polynucleotide designed to encode a protein fragment. Proteinfragments include one or more portions of the protein, e.g. proteindomains or subdomains. In some embodiments, the polynucleotide is anengineered polynucleotide designed to encode a mutated MOMP. Inparticular, in some embodiments the polynucleotide can also be designedsuch that the resulting protein, protein fragment or mutated MOMP isexpressed as a fusion, or chimeric protein product (i.e. it is joinedvia a peptide bond to a heterologous protein sequence of a differentprotein), for example to facilitate purification or detection. Achimeric product can be made by ligating the appropriate nucleic acidsequences encoding the desired amino acid sequences to each other usingstandard methods and expressing the chimeric product. In particular, insome embodiments, the polynucleotide can be engineered so that the MOMPis labeled or tagged. Labeling or tagging can be performed with methodsthat include, for example, FRET pairs, NHS-labeling, fluorescent dyes,and biotin as well as coding for a “His-tag” to enable protein isolationand purification via established Ni-affinity chromatography.

In some embodiments herein described, the polynucleotide is a DNAmolecule that can be in a linear or circular form, and encodes thedesired polypeptide under the control of a promoter specific to anenzyme such as an RNA polymerase, that is capable of transcribing theencoded portion of the DNA.

In embodiments where the polynucleotide is DNA, the DNA may betranscribed as part of the cell free reactions or system. In thoseembodiments, the DNA contains appropriate regulatory elements, includingbut not limited to ribosome binding site, T7 promoter, and T7terminator, and the reagents or compounds include appropriate elementsfor both transcription and translation reactions. In other embodimentswhere the polynucleotide is RNA, the RNA can be prepared prior toaddition to the cell free reactions/system, wherein the polypeptide ofinterest is produced, and the reagents or compounds include appropriateelements for translation reactions only.

Accordingly, as used herein, the term “cell free expression”, “cell freetranslation”, “in vitro translation” or “IVT” refer to methods andsystems wherein the transcription and translation reactions are carriedout independently, and to systems in which the transcription andtranslation reactions are carried out simultaneously in a non-cellularcompartment, e.g. glass vial.

In each of these methods and systems, the reagents or compoundstypically include a cell extract capable of supporting in vitrotranscription and/or translation as appropriate. In any case, the cellextracts contain all the enzymes and factors to carry out the intendedreactions, and in addition, be supplemented with amino acids, an energyregenerating component (e.g. ATP), and cofactors, including factors andadditives that support the solubilization of the protein of interest.

These systems are known in the art and can be identified by the skilledperson upon reading of the present disclosure, and exist for botheukaryotic and prokaryotic applications. Exemplary cell free expressionsystems that can be used in connection with the methods and systems ofthe present disclosure includes but are not limited to commercial kitsfor various species such as extracts available from Invitrogen, Ambion,Qiagen and Roche Molecular Diagnostics, cellular extracts made from E.coli or wheat germ or rabbit reticulocytes, or prepared followingprotocols, such as published laboratory protocols, identifiable by askilled person upon reading of the present disclosure.

In some embodiments, the cell free system can operate in batch mode orin a continuous mode. In the batch mode, the reaction products remain inthe system and the starting materials are not continuously introduced.Therefore, in batch mode, the system produces a limited quantity ofprotein. In a continuous mode instead, the reaction products arecontinuously removed from the system, and the starting materials arecontinuously restored to improve the yield of the protein products andtherefore the system produces a significantly greater amount of product.

In some embodiments, MOMP-t-NLPs herein described can be assembled by atranslation method, where self-assembly of the NLPs can be achievedwhile the apolipoprotein or other scaffold protein is provided as aprotein in a mixture also comprising one or more membrane forminglipids, one or more telodendrimers, a polynucleotide coding for the MOMPand/or a MOMP fragment, and a scaffold protein. In some embodiments, thescaffold protein to telodendrimer mass ratio can be 15:1 to 1:1,preferably 5:1. In some embodiments, scaffold protein to lipids massratio can be 1.5:1 to 0.1:1, preferably 0.5:1. In some embodiments,scaffold protein to lipids mass ratio will be reduced when MOMP isinserted and may be altered to 1.5:0.75 to 0.1:0.75, preferably 0.5:0.75

In some embodiments, MOMP-t-NLPs herein described can be assembled by atranslation method, where self-assembly of the NLPs can be achievedwhile the apolipoprotein or other scaffold protein is being translatedfrom mRNA as described for example in [5-7]. In this process, expressionsystem lysates are mixed with the lipid and telodendrimer component ofthe NLP and plasmid DNA encoding the scaffold protein. The reaction canthen be allowed to proceed until assembly occurs during apolipoproteinexpression (e.g. for approximately 4-24 hrs). The apolipoproteintypically contains an affinity tag (e.g. His-tag) for subsequentpurification of the self-assembled NLP from the lysate.

In some embodiments, the ratio of lipid to telodendrimer to be addedduring the assembly process is 1:1 (W/W) to 1:100 (W/W). In someembodiments, the ratio of DNA encoding MOMP and/or a MOMP fragment toDNA encoding scaffolding protein is between 1:1 (W/W) to 200:1 (W/W).Preferably, the ratio of lipid to telodendrimer to be added during theassembly process is 10:1 (W/W). Preferably, the ratio of DNA encodingMOMP and/or a MOMP fragment to DNA encoding scaffolding protein isbetween 5:1 to 50:1, more preferably between 10:1 to 25:1.

In some embodiments, wherein the MOMP-NLP comprises a MOMP-fragment theratio of plasmids (pApo:pMOMP-fragment) can be varied in the cell freereaction to control the amount of fragmented MOMP made and insertedduring the assembly process. Normally, we use is 1:1 (W/W) to 1:250(W/W).

In some embodiments, telodendrimers concentrations can be optimized fora MOMP and/or a MOMP fragment by mixing them with lipids atconcentrations from 0.5-10 mg (telodendrimer) and 5-60 mg (lipid) permL. In some embodiments, the telodendrimer and lipid concentration canbe at a 2 mg (telodendrimer) and 20 mg (lipid) per mL prior to additionto the cell-free reaction. In some of those embodiments, the MOMP and/orMOMP fragment assembled in the NLPs form tertiary structures recognizedusing a conformational antibody, which has never been seen with otherrecombinant forms of MOMP.

In some embodiments, the methods and systems herein described areperformed at predefined lipid protein ratio, assembly conditions and/orwith the use of preselected protein component (formed by MOMP andScaffold protein as polynucleotide) and lipid component (formed by Lipidand telodendrimers) so as to increase the yield, control the size andcomposition of the resulting NLP, provide an NLP of pre-determineddimensions, achieve desired functionality of the NLP, such as a certainlevel of loading capacity for a target molecule. In some embodiments,the molar ratio of lipid component to scaffold protein component is10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 110:1,120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1, 190:1, 200:1, 210:1,220:1, 230:1, and 240:1. In NLPs herein described, the lipid to scaffoldprotein component ratio can be determined on a case by case basis inview of the experimental design as will be understood by a skilledperson.

In some embodiments, the scaffold protein is selected to define the sizeof empty NLPs. In particular, the scaffold protein and/or the membraneforming lipid can be selected so that the scaffold protein and themembrane forming lipid are contacted at a mass ratio of scaffold proteinto membrane forming lipid from about 1:10 to about 1:1 to provide aparticle having a size from 10 to 60 nm. In some embodiments, LipophorinIII lipoproteins may assemble into larger NLPs with diameters 10-30 nmrange, apolipoprotein A1 NLPs range in size from 10-25 nm, truncated Δ(1-49) Apolipoprotein A1 15-35 nm. Adjustment of protein to lipid ratiosby increasing lipid will also increase the size of the NLP. Anexemplary, procedure is illustrated in the examples section. Inclusionof MOMP protein can cause up to 4-fold increase in size to thedimensions of an empty NLP.

In some embodiments, the method to assemble MOMP-t-NLPs herein describedresults in increasing MOMP to achieve detectable MOMP solubility. Inparticular, solubility can be measured by centrifuging the totalcell-free mixtures following completion of the cell free reaction (e.g.by a table centrifuge at max speed for 10 minutes). Aftercentrifugation, the supernatant is collected and MOMP solubilitycalculated as ratio of the amount MOMP protein in supernatant to theamount of MOMP protein in the total mixture. A percentage solubility canbe the calculated by calculating the amount of the MOMP present in thesupernatant (e.g. in term of molar concentration or mass concentration.

In particular, in some embodiments, a detectable solubility forrecombinant or native MOMP can be achieved without the need of addingdetergents. In particular, in some embodiments, no exogenously addeddetergent is required for solubilization of MOMP to a detectablesolubility. In some embodiments, the increased levels of MOMP solubilityare detectable with formation of NLPs within the cell free reaction.

In some embodiments, the method to assemble MOMP-t-NLPs herein describedresults into as high as 8:1 MOMP membrane protein and/or MOMP fragmentinsertion ratio to NLP, which has not been achieved previously.

In particular in some embodiments, wherein the MOMP-NLP comprises MOMPfragment, the NLP to MOMP ratio is expected to be between 1:1 and 1:25(Apo:MOMP protein) depending on the fragment size.

In some embodiments, the method to assemble MOMP-t-NLPs herein describedresults in a MOMP protein and/or a MOMP fragment with a greater than 2mg/mL of >85% purity without need of using affinity or solubilizationtags directly encoded on the MOMP protein.

In some embodiments, the formulation resulting from method to assembleMOMP-t-NLPs herein described in a form susceptible to lyophilization andresolubilization, which result in MOMP-tNLPs that were intact andfunctional. Accordingly, in some embodiments, the method to assembleMOMP-t-NLPs herein described, the combination of the materials usedtherein provide stabilized MOMP-t-NLPs.

In some embodiments, any of the MOMP-t-NLP herein described can becomprised in a composition together with a suitable vehicle. The term“vehicle” as used herein indicates any of various media acting usuallyas solvents, carriers, binders or diluents of a MOMP-t-NLP comprised inthe composition as an active ingredient.

In some embodiments, the composition of the disclosure comprises a sametype of MOMP-t-NLP. In some embodiments, the composition can comprisemore than one type of MOMP-t-NLP presenting different combination ofMOMP, MOMP fragments, Pmps, adjuvants and/or other components, and/orpresenting a same or different combination of MOMP, MOMP fragments,Pmps, adjuvants and/or other components at different ratios, as will beunderstood by a skilled person.

In some embodiments, MOMP-t-NLP can be included in pharmaceuticalcompositions (e.g. a vaccine) together with an excipient or diluent. Inparticular, in some embodiments, pharmaceutical compositions aredescribed which contain MOMP-t-NLP, in combination with one or morecompatible and pharmaceutically acceptable vehicle, and in particularwith pharmaceutically acceptable diluents or excipients.

The term “excipient” as used herein indicates an inactive substance usedas a carrier for the active ingredients of a medication. Suitableexcipients for the pharmaceutical compositions herein disclosed includeany substance that enhances the ability of the body of an individual toabsorb the NLP. Suitable excipients also include any substance that canbe used to bulk up formulations with NLP to allow for convenient andaccurate dosage. In addition to their use in the single-dosage quantity,excipients can be used in the manufacturing process to aid in thehandling of NLP. Depending on the route of administration, and form ofmedication, different excipients may be used. Exemplary excipientsinclude but are not limited to antiadherents, binders, coatingsdisintegrants, fillers, flavors (such as sweeteners) and colors,glidants, lubricants, preservatives, sorbents.

The term “diluent” as used herein indicates a diluting agent which isissued to dilute or carry an active ingredient of a composition.Suitable diluents include any substance that can decrease the viscosityof a medicinal preparation.

In certain embodiments, compositions and, in particular, pharmaceuticalcompositions can be formulated for systemic administration, whichincludes parenteral administration and more particularly intravenous,intradermic, and intramuscular administration. In some embodiments,compositions and, in particular, pharmaceutical compositions can beformulated for non-parenteral administration and more particularlyintranasal, intratracheal, vaginal, oral, and sublingual administration.

In some embodiments, the compositions herein described are administratedto humans or animals via mucosal vaccination routes. The compositionscan be delivered to a subject through oral mucosa or intranasalinhalation, allowing a direct absorption into the systemic circulation.

Exemplary compositions for parenteral administration include but are notlimited to sterile aqueous solutions, injectable solutions orsuspensions including MOMP-t-NLPs. In some embodiments, a compositionfor parenteral administration can be prepared at the time of use bydissolving a powdered composition, previously prepared in a freeze-driedlyophilized form, in a biologically compatible aqueous liquid (distilledwater, physiological solution or other aqueous solution).

The term “lyophilization” (also known as freeze-drying orcryodesiccation) indicates a dehydration process typically used topreserve a perishable material or make the material more convenient fortransport. Freeze-drying works by freezing the material and thenreducing the surrounding pressure and adding enough heat to allow thefrozen water in the material to sublime directly from the solid phase togas.

If a freeze-dried substance is sealed to prevent the reabsorption ofmoisture, the substance may be stored at room temperature withoutrefrigeration, and be protected against spoilage for many years.Preservation is possible because the greatly reduced water contentinhibits the action of microorganisms and enzymes that would normallyspoil or degrade the substance.

Lyophilization can also cause less damage to the substance than otherdehydration methods using higher temperatures. Freeze-drying does notusually cause shrinkage or toughening of the material being dried. Inaddition, flavours and smells generally remain unchanged, making theprocess popular for preserving food. However, water is not the onlychemical capable of sublimation, and the loss of other volatilecompounds such as acetic acid (vinegar) and alcohols can yieldundesirable results.

Freeze-dried products can be rehydrated (reconstituted) much morequickly and easily because the process leaves microscopic pores. Thepores are created by the ice crystals that sublimate, leaving gaps orpores in their place. This is especially important when it comes topharmaceutical uses. Lyophilization can also be used to increase theshelf life of some pharmaceuticals for many years.

In pharmaceutical applications freeze-drying is often used to increasethe shelf life of products, such as vaccines and other injectables. Byremoving the water from the material and sealing the material in a vial,the material can be easily stored, shipped, and later reconstituted toits original form for injection.

In some embodiments, MOMP-t-NLPs herein described can be used as animmunostimulatory particle and in particular as immunostimulatoryparticles directed to obtain an immunitary response against one or morebacteria of the genus Chlamydia.

The term immunostimulatory as used herein describes the stimulation ofthe immune system and in particular the ability of a compound, complexand/or particle to affect the immune system.

The immunostimulatory MOMP-t-NLPs herein described are configured topresent MOMP as an immunological agent on the t-NLP alone or togetherwith other immunological agents such as other antigens or single ormultiple adjuvants. In preferred embodiments theimmunostimulatory-MOMP-t-NLPs herein described comprise MOMPS primarilyforming homo-trimers in effective amount to elicit an immunologicalresponse [3].

The term “immunological agent” as used herein indicates a compound thatis able to interfere with the immune system of an individual, and inparticular provoke, reduce, enhance or impair a response of the immunesystem under same or comparable conditions. Exemplary immunologicalagents comprise antigen and adjuvants.

The term “antigen” or “immunogen” as used herein indicates a substancethat prompts the generation of antibodies and/or can cause an immuneresponse. In particular, antigens in the sense of the present disclosureencompass all substances that can be recognized by an adaptive immunesystem. Exemplary antigens include exogenous antigens and endogenousantigens. Exogenous antigens are antigens that have entered the bodyfrom the outside, for example by inhalation, ingestion, or injection. Byendocytosis or phagocytosis, these antigens are taken into theantigen-presenting cells (APCs) and processed into fragments. APCs thenpresent the fragments to T helper cells (CD4⁺) by the use of class IIhistocompatibility molecules on their surface. Some T cells are specificfor the peptide: MHC complex. They become activated and start to secretecytokines. Cytokines are substances that can activate cytotoxic Tlymphocytes (CTL), antibody-secreting B cells, macrophages, and otherparticles. Endogenous antigens are antigens that have been generatedwithin the cell, as a result of normal cell metabolism, or because ofviral or intracellular bacterial infection or transformation of cellsleading to cancer. The fragments are then presented on the cell surfacein the complex with MHC class I molecules. If activated cytotoxic CD8⁺ Tcells recognize them, the T cells begin to secrete various toxins thatcause the lysis or apoptosis of the infected cell. In order to keep thecytotoxic cells from killing cells just for presenting self-proteins,self-reactive T cells are deleted from the repertoire as a result oftolerance (also known as negative selection). They include xenogenic(heterologous), autologous and idiotypic or allogenic (homologous)antigens. Antigens are also generated between normal cells.

In some embodiments, the immunostimulatory MOMP-t-NLPs herein describedcomprise an immunogenic fragment of the MOMP protein or MOMP immunogenicfragment. The term “immunogenic fragment” as used herein refers to afragment of a protein that is capable of eliciting a specific immuneresponse, such as an epitope for a B-cell or T-cell as will beunderstood by a skilled person. In particular, in embodiments hereindescribed a MOMP immunogenic fragment is a MOMP fragment in the sense ofthe disclosure comprising an immunogenic region including immunogenicMOMP domains in addition to a transmembrane region comprising MOMPhydrophobic amino acid configured to interact with a membrane lipidbilayer. In particular, in a MOMP immunogenic fragment the immunogenicregion can be formed by one or more of the variable domains and/or oneor more of the epitopes of the MOMP protein, having 1 to 100 amino acidresidues each.

Reference is made in this connection to the illustration of FIG. 1A,which shows exemplary variable domains (residues circled) and exemplarytransmembrane domains (residues within squares) of MOMP protein. Inparticular FIG. 1A shows Protein sequence of major outer membraneprotein (MOMP), Chlamydia tracomatis, serovar CLPVGNPAEPSLMIDGILWEGFGGDPCDPCTTWCDAISMRVGYYGDFVFDRVLKTDVNKEFQMGAAPTTSDVAGLQNDPTINVARPNPAYGKHMQDAEMFTNAAYMALNIWDRFDVFCTLGATTGYLKGNSASFNLVGLFGTKTQSSSFNTAKLIPNTALNEAVVELYINTTFAWSVGARAALWECGCATLGASFQYAQSKPKVEELNVLCNASEFTINKPKGYVGAEFPLNITAGTEAATGTKDASIDYHEWQASLALSYRLNMFTPYIGVKWSRVSFDADTIRIAQPKLAEAILDVTTLNRTTAGKGSVVSAGTDNELADTMQIVSLQLNKMKSRKSCGIAVGTTIVDADKYAVTVEARLIDERAAHVNAQFRF (SEQ ID NO: 66)

Reference is also made to the illustration of FIGS. 1B and 1C showingMOMP variable domains (herein also VD) in schematic illustration of MOMPtridimensional structure from Feher et al. [1] 2013 (see FIG. 4)incorporated herein by reference in its entirety.

Reference is also made to the illustration of FIG. 1D showing a MOMPmultiepitope described in Tu et al. 2013 [2] incorporated herein byreference in its entirety; the MOMP multiepitope is expected to becomprised in MOMP immunogenic fragments herein described fused to a MOMPtransmembrane region in the sense of the disclosure.

The term “epitope” as used herein, also known as an “antigenicdeterminant” refers to the part of an antigen that is recognized by theimmune system, specifically by antibodies, B cells, or T cells. Forexample, the epitope is the specific piece of the antigen to which anantibody binds. The part of an antibody that binds to the epitope iscalled a paratope. Although epitopes are usually non-self proteins,sequences derived from the host that can be recognized (as in the caseof autoimmune diseases) are also epitopes.

As a person skilled in the art would understand, the epitopes of proteinantigens are divided into two categories, comprising conformationalepitopes and linear epitopes, based on their structure and interactionwith the paratope. A conformational epitope is composed of discontinuoussections of the antigen's amino acid sequence. These epitopes interactwith the paratope based on the 3-D surface features and shape ortertiary structure of the antigen. By contrast, linear epitopes interactwith the paratope based on their primary structure. A linear epitope isformed by a continuous sequence of amino acids from the antigen.

For example, T cell epitopes are presented on the surface of anantigen-presenting cell, where they are bound to MHC molecules. Inhumans, antigen-presenting cells are specialized to present MHC class IIpeptides, whereas most nucleated somatic cells present MHC class Ipeptides. T cell epitopes presented by MHC class I molecules aretypically peptides between 8 and 11 amino acids in length, whereas MHCclass II molecules present longer peptides, 13-17 amino acids in length,and non-classical MHC molecules also present non-peptidic epitopes suchas glycolipids.

Epitopes can be mapped, for example using protein microarrays, or withELISA or ELISPOT techniques, among others known to those skilled in theart. Another technique involves high-throughput mutagenesis, an epitopemapping strategy developed to improve rapid mapping of conformationalepitopes on structurally complex proteins [8]. In addition, MHC class Iand II epitopes can be predicted by computational means [9]. Additionalmethods for identifying epitopes are described in U.S. Pat. Nos.8,889,142, 8,486,411, 7,754,228 and 6,635,746 and will be understood bya person skilled in the art.

In particular, an immunogenic fragment of MOMP refers to a fragment of aMOMP protein that is capable of eliciting a Chlamydia specific immuneresponse in a host. As would be understood by persons skilled in theart, an immune response in a host is typically mediated by recognitionby the host immune system of a specific protein epitope.

Mapping of MOMP B-cell epitopes recognized by antibodies elicited byimmunization can be performed following techniques known in the art,such as those described in Tifrea et al. 2014 [10]. Examples of MOMPimmunogenic epitopes comprise those of epitopes within MOMP variabledomains (VD) VD1, VD2, or VD4, or constant domain (CD) CD2, CD3, CD4, orCD5; the sequences of oligomer peptide probes used to detect theepitopes and the corresponding MOMP protein domains recognized are shownin Table 2.

TABLE2 Oligomer MOMP probe domain C. muridarum MOMP amino acid sequenceSEQ ID NO P5 VD1 EMGAAPTGDADLTTAPTPASRENPA  7 P6 VD1PTPASRENPATGKHMQDAEMFTNAA  8 P10 VD2 FGRDETAVAADDIPNVSLSQAVVEL  9 P20VD4 TSILKMTTWNPTISGSGIDVDTKIT 10 P4 VD1 FVFDRVLKTDVNKQFEMGAAPTGDA 11 P9VD2 GYLKGNSAAFNLVGLFGRDETAVAA 12 P7 CD2 QDAEMFTNAAYMALNIWDRFDVFCT 13 P23CD5 LAIGTTIVDADKYAVTVETRLIDER 14 P24 CD5 TVETRLIDERAAHVNAQFRF 15 P14 CD3KVEELNVLCNAAEFTINKPKGYVGQ 16 P19 Includes SFDADTIRIAQPXLETSILKMTTWN 17regions of CD4 and VD4 P21 Overlaps SGIDVDTKITDTLQIVSLQLNKMKS 18VD4 and CD5 P22 CD5 VSLQLNKMKSRKSCGLAIGTTIVDA 19

Additional exemplary immunogenic MOMP peptide fragments and epitopes ofC. trachomatis are identifiable by those skilled in the art, inpublished articles such as in [2, 11-14], among others, and such asthose described in U.S. Pat. Nos. 8,889,142, 8,486,411, 7,754,228 and6,635,746.

Identification of MOMP epitopes can also be determined in part byanalysis of structure of MOMP proteins. Exposed domains of MOMP areunderstood to be both serotyping and protective antigenic determinants[12]. The four topological models of MOMP, corresponding to C. muridarumand the C. trachomatis serovars C, D and F, have been proposed and canbe used in the protein structural analysis [1, 15-17].

The immunostimulatory MOMP-t-NLP herein described can further compriseadjuvants.

The term “adjuvant” as used herein indicates an agent that stimulatesthe immune system but that is not antigenic in itself. Typically,adjuvants are used in connection with antigens and/or vaccinecomposition to increase the response to one or more antigen of choice.

Exemplary adjuvants that can be incorporated into an NLP hereindescribed as a self-assembling component comprise; naturally occurringhydrophobic or amphipathic adjuvants, including but not limited tolipopolysaccharides (LPS), mono-phosphorylated Lipid A (MPLA), organiccompounds (squalene, sorbitol oleate esters), alpha-galactosyl ceramide,and lipoteichoic acid (LTA), or hydrophilic adjuvants syntheticallyappended with a hydrophobic moiety, including for example microbialderivatives (e.g. CpG motifs, muramyl dipeptide (MDP), flagellin), plantderivatives (e.g. saponins), and immunostimulatory proteins (e.g.cytokines, toxins, and derivative peptides), and immunostimulatorycarbohydrates and polysaccharides.

In particular, in some embodiments, immunostimulatory MOMP-t-NLP hereindescribed present MPLA alone or in combination with additionaladjuvants. MLPA is a well-established adjuvant that has been shown toinduce both cellular and humoral immune responses. MPLA is a lowtoxicity derivative of a bacterial cell wall component,lipopolysaccharide (LPS).

In any of the above embodiments, one or more additional same ordifferent adjuvant and/or antigen can be attached to theimmunostimulatory MOMP-t-NLPs through binding the anchor compound-anchorsubstrate compound and/or through incorporation of an amphipathicadjuvant into the nanoparticle during self-assembly.

In some embodiments, binding or conjugation of the adjuvant or otherimmunological agent can be performed by chelation of the immunologicalagent to a functional group presented by one or more functionalizedlipids in the MOMP-t-NLPs herein described. The term “chelation” as usedherein indicates the binding or complexation of a bi- or multidentateligand with a single metal ion. In particular, in some embodiments, thebi or multi-dentate ligand is part of the lipid and is capable ofbinding a metal ion. The ligands, which are often organic compounds, arecalled chelants, chelators, chelating agents, or sequestering agents.Chelating agents form multiple bonds with a single metal ion. The term“chelants” as used herein indicates a molecule that forms a stablecomplex with certain metal ions. Examples of chelating moieties include,but are not limited to, nitrilotriaceticacid (NTA), iminodiacetic acid(IDA), and diethylenetriamine penta-acetic acid (DTPA).

Successful binding of an immunological agent to the NLP can be readilyverified and quantified through a range of techniques that include butare not limited to centrifugal filtration, size exclusionchromatography, fluorescence correlation spectroscopy, cantilever-basedsensing, force spectroscopy, Fourier transform infrared spectroscopy,surface plasmon resonance, total internal reflection fluorescence, Ramanspectroscopy and additional techniques identifiable by a skilled person.In addition, binding specifically to the surface can be verified usingatomic force microscopy and transmission electron microscopy andadditional techniques identifiable by a skilled person.

In some embodiments, the formation of immunostimulatory MOMP-t-NLPsherein described is amenable to the incorporation of multiple adjuvants,including for example compounds directed to enhance immune response e.g.non-human lipoproteins, bacterial peptides, DNA (e.g. CpG motifs),chemokines, cytokines, pattern-recognition receptors (PRR), lipids,polysaccharides, lipopolysaccharides, and the like; in general, agonistsand immune stimulatory molecules, synthetic or natural, (known orunknown at this time) can be assembled in or on NLPs, providing forenhanced, specific, rapid immune stimulation at the site of NLP/antigeninoculation and spreading systemically.

In some embodiments, the formulated MOMP t-NLPs with single or multipleadjuvant result in a sustained IgG titer that are several logs higherthan adjuvant-NLPs or NLPs alone. Adjuvants concentrations can be variedup to 20 μg per dose. In preferred embodiments, MOMP t-NLPs can comprisetwo or more adjuvants to provide MOMP t-NLPs capable of eliciting anoptimal protective response with MOMP.

In some embodiments, immunostimulatory MOMP-t-NLPs herein described canbe comprised of immunostimulatory compositions, including vaccines to beadministered to individuals.

The term “individual” as used herein in the context of treatmentincludes a single biological organism, including but not limited to,animals and in particular higher animals and in particular vertebratessuch as mammals and in particular human beings

The immunostimulatory MOMP-t-NLPs or the immunostimulatory compositionherein described can also be administered to an individual alone or incombination with additional immunostimulatory agents to immunize theindividual.

In particular, in some embodiments, MOMP-t-NLPs herein described can beused in combination with NLPs comprising an adjuvant such as microbialderivatives (e.g. CpG derivatives, MPLA), muramyl dipeptide derivatives(e.g. muroctasin), and any peptide or protein adjuvants (e.g. flagellin)can be incorporated into NLP directly to create an adjuvant NLP that canbe used as an adjuvant or as a platform for subunit vaccine developmentwith enhanced potency.

In particular, an adjuvant NLP according to the present disclosure cancomprise single or multiple adjuvants, such as CpGs, MPLA, andcytokines. In some embodiments, an adjuvant NLP can be customized byincluding for example selected adjuvants in view of the desired effectbased on the ability of different adjuvants to target differenttoll-like receptors (TLR) for immunostimulation (e.g. MPLA targets TLR4, CpGs target TLR9, and flagellin targets TLR5). In some of theseembodiments, the customization is performed in view of a specificvaccine formulation to be used in combination with the adjuvant NLP. Thecustomization can be made to combine in the NLP only the adjuvants thatare effective for the vaccine formulation of choice, since in somevaccine formulations only certain adjuvants are successful at enhancingthe efficacy of the vaccine.

In some embodiments, the MOMP-t-NLP herein described are provided in aformulation compatible with intramuscular or intranasal administrationin an amount effective to elicit a protective response. In someembodiments, the MOMP-t-NLP herein described are provided in aformulation for intravaginal administration in an amount effective toelicit a protective response by vaginal exposure to a Chlamydiapathogen.

Immunization can be affected by simple intramuscular injection in eitherthe shoulder area or in the gluteus maximus hind muscular region.Particles could be delivered following solubilization in sterile normalsaline solution, for example. Such immunizations would be subject topractices and methods approved by the US government Food and DrugAdministration (FDA).

In particular, in some embodiments, the immunostimulatory NLPs thatcomprise at least one antigen can be used as vaccines that can beprepared rapidly and are relatively stable affording the desiredprotective immune response in accordance with attached immunogen.

The term “vaccine” as used herein indicates a composition, and inparticular a biological preparation, that establishes or improvesimmunity to a particular external pathogenic assault, or an inherenttransformational incident resulting in a cancerous or autoimmunecondition in mammals. Vaccines in the sense of the present descriptioncan be prophylactic, or therapeutic.

In some embodiments, the immunostimulatory MOMP-t-NLP construct is moreimmunogenic than the antigen alone, and can be used as a vaccine toprotect against Chlamydia infection when injected into an appropriaterecipient with or without the aid or use of an adjuvant type carrier.

In particular, in some embodiments, methods herein described allowproduction of a functional MOMP protein in immunostimulatory MOMP-t-NLPsfor vaccine development despite MOMP poor solubility, low yield, andprotein misfolding which characterize MOMP production thus provideimmunogenic MOMP or fragment thereof in particular in the preferred andmost preferred embodiments herein described as will be understood by askilled person upon reading of the present disclosure. The dimension andcomplexity of MOMP renders it difficult to recombinantly synthesize in acorrectly folded state. For example, efforts to express MOMP inbacterial systems have yielded poor results due to incorrect MOMPprotein folding [15, 18, 19]. In addition, processes of extractingnative MOMP from Chlamydia is laborious and is difficult to produce forlarge-scale commercial applications. Experimental MOMP vaccines based ondenatured or non-native recombinant preparations have shown to yieldonly partial protection in a mouse model using C. muridarum [10, 20-22].

The cell-free expression methods and systems described herein canproduce a MOMP-tNLP complex with the tNLP membrane-bound MOMP formingmultimers similar to the native protein in high yield, with increasedsolubility (Example 2), and retained functionality and immunogenicity(Examples 5-7), while eliminating the need to overexpress insoluble MOMPproteins in cells or to reconstitute MOMP with detergent. The processdescribed herein can also be applied to other membrane-bound proteinspreviously difficult to obtain antigens in vivo or difficult to producein native higher order structures.

In several embodiments, the immunostimulatory MOMP-t-NLP presentingantigens alone or in combination with adjuvants conjugates encapsulatekey requirements for vaccine formulation: non-virulence;immunostimulation; clustered antigen presentation; expression of MOMP inmultimeric form required for effective immune response; simple, rapid,inexpensive production; and the means to accommodate a wide range ofselect-agent antigens. Furthermore, adjuvant-bearing NLPs promote bothhumoral and cellular immune responses.

In some embodiments, the immunostimulatory MOMP-t-NLP presentingantigens alone or in combination with adjuvants in a vaccine fortreating or preventing a Chlamydia infection or conditions associatedthereto via intramuscular or intranasal administration. In someembodiments, the immunostimulatory MOMP-t-NLP presenting antigens aloneor in combination with adjuvants in a vaccine for treating or preventinga Chlamydia infection or conditions associated thereto via intravaginaladministration.

In several embodiments, the immunostimulatory MOMP-t-NLP are hereindescribed and related compositions, methods and systems allow costeffective and rapid development of immunostimulatory compositions thatare safe, enable immunization with multivalent/or broad-spectrumresponse and at the same time, are able to elicit a high levelsprotection following an adequate stimulation of a host immune response.

In several embodiments, the immunostimulatory MOMP-t-NLP, methods andsystems herein described allow incorporation in the immunogenicparticles of secondary additives to enhance immune response in theindividual.

In certain embodiments, an adjuvant and one or more immunostimulatoryMOMP-t-NLP can also be comprised in a system to immunize an individual.In those embodiments, the system comprises: the immunostimulatoryparticle herein described and an adjuvant, the immunostimulatoryparticle and the adjuvant to be administered to the individual toimmunize such individual.

The systems herein disclosed can be provided in the form of kits ofparts. In kit of parts for the production of MOMP-t-NLPs hereindescribed, the MOMP and the scaffold protein can be included in the kitas a protein alone or in the presence of lipids/detergents fortransition into nanoparticles. The MOMP and/or the scaffold protein canbe included as a plasmid or PCR DNA product fortranscription/translation. The indicator protein may be included asencoded RNA for translation. In kit of parts for the immunization of anindividual the immunostimulatory MOMP-t-NLP can be comprised togetherwith adjuvant and/or adjuvant NLPs and additional componentsidentifiable by a skilled person.

In a kit of parts, a polynucleotide, amphipathic lipid, target proteinand/or scaffold protein, MOMP-tNLPs, adjuvants, adjuvant NLPs andadditional reagents are comprised in the kit independently possiblyincluded in a composition together with suitable vehicle carrier orauxiliary agents. For example, a polynucleotide can be included in oneor more compositions alone and/or included in a suitable vector, andeach polynucleotide in a composition together with a suitable vehiclecarrier or auxiliary agent. Furthermore, the target protein can beincluded in various forms suitable for appropriate incorporation intothe NPL.

Additional components can include labeled polynucleotides, labeledantibodies, labels, microfluidic chip, reference standards, andadditional components identifiable by a skilled person upon reading ofthe present disclosure. In particular, the components of the kit can beprovided, with suitable instructions and other necessary reagents, inorder to perform the methods here disclosed. The kit will normallycontain the compositions in separate containers. Instructions, forexample written or audio instructions, on paper or electronic supportsuch as tapes or CD-ROMs, for carrying out the assay, will usually beincluded in the kit. The kit can also contain, depending on theparticular method used, other packaged reagents and materials (i.e. washbuffers and the like).

Further details concerning the identification of the suitable carrieragent or auxiliary agent of the compositions, and generallymanufacturing and packaging of the kit, can be identified by the personskilled in the art upon reading of the present disclosure.

EXAMPLES

The methods and system herein disclosed are further illustrated in thefollowing examples, which are provided by way of illustration and arenot intended to be limiting.

In particular, mMOMP-tNLPs comprising a MoPn MOMP protein (mMOMP, a typeof MOMP expressed in the mouse-specific Chlamydia muridarum), scaffoldprotein Δ49apolipoprotein A1 (Δ49ApoA1, a truncated version of mouseApoA1), membrane-forming lipids, and telodendrimers were prepared usinga cell-free expression system and characterized in vitro and in vivo.The mMOMP-tNLP particle also accommodated the co-localization of the CpGadjuvant ODN1826 for in vivo characterization. A skilled person will beable to use the membrane forming lipids, telo-dendrimers, scaffoldproteins, adjuvant, and mMOMP herein described. The following materialsand methods were used.

Plasmids: The truncated form of mouse Apo A1 (A1-49) or Δ49ApoA1 geneand mMOMP gene were assembled from oligonucleotides and cloned intoNdeI/BamHI digested pIVEX2.4d vector (Roche Molecular Diagnostics,Basel, Switzerland) using Gibson Assembly. Briefly, Archetype Softwarewas used to design 60 bp long, overlapping oligonucleotides covering theDNA sequence of interest (Δ49ApoA1 including 90 bp 5′ and 3′ vectoroverlap to pIVEX2.4d). The 60 bp oligonucleotides overlapped neighboringoligonucleotides by 30 bp. In addition, forward and reverse primers(distal primers) were designed for amplification of the DNA sequence ofinterest. The pIVEX2.4d vector contained a His-tag used for nickelaffinity purification as previously described [23]. The codon-optimizedplasmid sequences are shown (FIGS. 3A-3B; also shown in FIGS. 12A-C, 13,14, 15, 16 and 17).

DMPC/telodendrimer preparation: PEG^(5k)-CA₈ telodendrimer was preparedaccording to a published method [24]. Small unilamellar vesicles of DMPC(Avanti Polar Lipids, Alabaster, Ala.) were prepared by probe sonicationof a 20 mg/mL aqueous solution of DMPC until optical clarity wasachieved; typically 3 intervals of 30 seconds were sufficient. After thesonication, the samples were centrifuged at 14,100 rcf for 1 minute toremove metal contamination from the probe tip. For the DMPC/PEG^(5k)-CA₈mixtures, a total of 20 mg/mL DMPC and 2 mg/mL PEG^(5k)-CA₈ were mixedat a volume ratio of 1:1.

Cell-free Reaction: Small and large scale reactions (50 μL and 1 mL)were carried out using RTS 500 ProteoMaster E. coli HY Kit(Biotechrabbit GmbH, Hannover, Germany). Small scale reactions containedthe same ratio of components as the large-scale reactions. Reactioncomponents (lysate, reaction mix, feeding mix, amino acid mix, andmethionine) were combined as specified by the manufacturer. Forexpression, 0.3-1.5 μg of Δ49ApoA1 and 15 μg mMOMP plasmid DNA was addedto each 1 mL reaction. A total of 400 μL DMPC/telodendrimer mixture wasthen added. The reactions were incubated at 30° C., with shaking at 300rpm for 14-18 hrs in a floor shaker.

Affinity purification of NLP-related complexes: Immobilized nickelaffinity chromatography was used to isolate the mMOMP-tNLP from thecell-free reaction mixture. 1 mL of 50% slurry cOmplete His-TagPurification Resin (Roche Molecular Diagnostics, Basel, Switzerland) wasequilibrated with equilibration buffer (50 mM NaH₂PO₄, 300 mM NaCl, pH8.0) with 10 mM imidazole (Sigma-Aldrich, St Louis, Mo.) in a 10 mLchromatography column. The total cell free reaction (1 mL) was mixedwith the equilibrated resin, and was incubated/mutated at 4° C. for 1hr. The column was then washed with equilibration buffer containing 20mM imidazole. The column was washed with 1 mL of the same buffer 6times. The mMOMP-tNLPs were eluted in six 300 μl fractions ofequilibration buffer containing 250 mM imidazole and 1 final elution of300 μl in 500 mM imidazole. All elutions were analyzed by SDS-PAGE andpeak fractions containing protein were combined. Pooled fractions weredialyzed in PBS (pH 7.4) and then stored at 4° C. Material for mousestudies were tested for endotoxin levels using the Endosafe-PTS (CharlesRiver, Charleston, S.C.) endotoxin testing system based on LimulusAmebocyte Lysate (LAL) assay. All NLP preparations have an endotoxinlevel between 20 and 100 EU/mg.

Size exclusion chromatography (SEC): NLPs were purified by SEC (Superdex200, 10/300 GL column, GE Healthcare, Piscataway, N.J.). SEC was run ata flow rate of 1 mL/min in PBS buffer with 0.25% PEG2000.

SDS PAGE: A total of 5-15 μL aliquots of the eluted mMOMP-tNLPs weremixed with 4× NuPAGE LDS Sample buffer and 10× NuPAGE Sample ReducingAgent (Life Technologies Corporation, Carlsbad, Calif.), heat denaturedand loaded onto a 4-12% gradient pre-made 1.0 mm Bis-Tris gel (LifeTechnologies Corporation, Carlsbad, Calif.) along with the molecularweight standard SeeBlue Plus2 (Life Technologies Corporation, Carlsbad,Calif.). The running buffer was 1×MES-SDS (Life TechnologiesCorporation, Carlsbad, Calif.). Samples were run for 35 minutes at 200V.Gels were stained with SYPRO Ruby Protein Gel stain (Life TechnologiesCorporation, Carlsbad, Calif.) according to manufacturer's instructions,and imaged using a LiCor Odyssey Fc Imager (LI-COR Biotechnology,Lincoln, Nebr.).

Western blots and dot blots analysis: Western and dot blots wereperformed on PVDF membranes (Millipore). For western blots, samples wereresolved with SDS-PAGE as described above. The gels were incubated intransfer buffer for 10 minutes and transferred at 4° C. for 65 minutesat 100V. The transfer buffer was 1×NuPAGE (Life TechnologiesCorporation, Carlsbad, Calif.). Blots were incubated overnight at 4° C.in Odyssey Blocking Buffer (PBS) (LiCor Biotechnology, Lincoln, Nebr.)containing 0.2% Tween-20 and either 0.5 mg/mL mAb40 (linear, VD1) or 0.2mg/mL Penta-His antibody (Qiagen, Hilden, Germany) diluted 1:1000 [25].Blots were then washed for five minutes, four times, with PBS-T (50 mMNaH₂PO₄, 300 mM NaCl, 0.2% Tween-20, pH 7.4) while shaking. Blots werethen incubated for 1 hour in blocking buffer containing 0.2% Tween-20,0.02% SDS and 1 mg/mL IRDye 800CW Goat (polyclonal) anti-Mouse IgG (H+L)(LI-COR Biosciences, Lincoln, Nebr.) diluted to 1:10,000. Blots werewashed with PBS-T four more times and imaged with LiCor Fc Imager at 800nm. For dot blots, 3 μg of purified nanoparticles with and without mMOMPwere blotted using the Bio-Dot Apparatus #1706545 (Bio-Rad), accordingto manufacturer's instructions. Blots were developed as mentioned above.

Conductance assays: To look at the ability of mMOMP to form functionalpores, the mMOMP-tNLP complex was incorporated into planar lipid bilayerand conductance measurements were performed in a two-chamber black lipidmembranes (BLM) cell (Eastern Scientific LLC, Rockville, Md., USA). Asupported DMPC lipid bilayer was formed over a 200 μm diameter aperturein a Teflon film partition using a painting technique. The cis-chamber(connected to ground Ag/AgCl electrode) and trans-chamber (connected toa reference Ag/AgCl electrode) were filled with 0.2 mL and 2 mL PBSbuffer (w/Mg²⁺ and Ca²⁺, pH 7.4) respectively. 1-2 μL mMOMP-tNLP complexin solution was added to the cis-chamber above the DMPC bilayer. Aholding potential between −100 mV to +100 mV was applied to thereference electrode, and the transmembrane current signal was recordedby the Axiopatch 200B patch clamp amplifier (Axon Instruments, Milpitas,Calif., USA) connected to a computer system running Clampex 10.3software (Axon Instruments). The current traces were acquired at asampling frequency of 10 kHz-100 kHz. The data were exported andanalyzed using PClamp 10.3 software (Axon Instruments) and Igor Pro 6.31(Wavemetrics Inc.).

Dynamic light scattering (DLS): Dynamic light scattering measurements ofthe NLP size were performed on a Zetasizer Nano ZS90 (MalvernInstruments, Malvern United Kingdom)) following the manufacturer'sprotocols. Each data point represents an average of at least 10individual runs.

Atomic Force Microscopy (AFM): AFM is a technique known to a skilledperson to investigate NLPs and membrane protein insertion¹⁴⁵⁻¹⁴⁸.Briefly, atomically flat mica disks are glued to metal substrates tosecure them to the scanner of a stand-alone MFP-3D AFM (Asylum Research,Santa Barbara, Calif.). Topographical images are obtained with“Biolevers” (Olympus, Tokyo, Japan) with a spring constant of 0.03 N/min a room temperature controlled room at 23+/−1° C. Images are taken inalternate contact (AC) mode in liquid, with very low amplitudes at theprimary resonance frequency that was obtained from thermal analysis ofthe cantilever in solution. Heights of features in images are determinedby histogram and statistical analysis as will be understood by a skilledperson 60,112,113.

Transmission Electron Microscopy (TEM): Samples are harvested using bothcontinuous carbon coated TEM grids and small silicon wafers with siliconnitride membranes (each ˜3 mm in diameter). For NLP samples, a 4 μL dropof the purified sample (0.5 mg/ml) can be adsorbed to a cleanedholey-carbon-coated copper EM grid, blotted with Whatman paper andrapidly plunge frozen. The resulting cryoEM grid can then be imagedusing low-dose exposure techniques on a JEOL JEM-2100F transmissionelectron microscope. Electron micrographs are direct images of thesample, acquiring a large dataset provides a statistical overview of thehomogeneity and aggregation of the protein or complex in solution¹⁴⁹⁻¹⁵¹

Cryo-electron microscopy (cryoEM): In cryoEM, a fully hydrated complexis frozen and then subjected to electron microscopy. This permits anadvantage in studying hydrated complexes used for detailed and accurateimage re-construction¹⁵²⁻¹⁵⁷ All tNLP and mMOMP-tNLP samples werepreserved as frozen hydrated specimen in the presence of saturatedammonium molybdate for scanning with a JEOL JEM-2100F transmissionelectron microscope (JOEL USA, Peabody, Mass.) at magnification of80,000× under liquid nitrogen temperature.

Mouse immune study: All animal studies were performed at LawrenceLivermore National Laboratory in PHS-assured facilities in accordancewith guidelines set by the Animal Care and Use Committee (IACUC). Female3-week old mice (BALB/c) were purchased from Jackson Laboratory (BarHarbor, Me.). Since 3-week old mice are pre-pubescent, they are moresusceptible to STI infection and more suitable than adult mice for theChlamydia studies. A total of 6 mice/group were vaccinated with thefollowing formulations: 1×10⁴ IFU's of EB obtained from Dr. Luis de laMaza at UC Irvine, 10 μg of tNLP with 5 μg CpG adjuvant, 10 μgmMOMP-tNLP plus 5 μg of CpG adjuvant, or PBS alone. Total volumes perinoculation were 50 μL. Animals were primed on day 1 and received boostsat days 21 and 42. Whole blood was drawn prior to each inoculation. Afinal bleed was conducted on day 61 post initial prime. Serum antigenspecific IgG antibody titers were measured using an enzyme-linkedimmunosorbent assay (ELISA). Immulon 2 HB microtiter plates (ThermoLabsystems, Franklin, Mass.) were coated with the appropriate antigen(200 ng/well), and then incubated with sera (2-fold serial dilutionsstarting at 1:100 dilutions) for 1 hour. Goat anti-mouse IgGHRP-conjugated antibody (KPL, Gaithersburg, Md.) was added to the platesfor 1 hour, and the bound HRP was detected by incubation with TMB(Sigma) quenched after 5 min with 1 M HCl. The reaction product wasquantitated by a spectrophotometer at 450 nm, and values were correctedfor background activity detected from wells that received diluent inplace of sera. The titration curves were then fit to a power function inMS Office Excel and titers were calculated from the fit function using acutoff absorbance value of the average background O.D.±3 S.D.

Example 1. Structural Characterization of Native MOMP

A structural characterization of MOMP was performed with TEM.

In particular, the TEM analysis performed shows monodispersed nativeMOMP (FIG. 23A) stained with 5% ammonium molybdate and placed oncontinuous carbon grids for observation. For model building, trimericMOMP particles are selected using a semi-automated particle selectiontool via EMAN 2.1 package [26] (FIG. 23B). The pre-processing of MOMPimage analysis shows that the protein is predominantly in a trimericassociation, with some sample heterogeneity

MOMP trimer images were then collected and class averaged. Theindividual MOMP trimer images were processed using reference-freeclassification to group particles with similar orientation (FIG. 23C).The images were then aligned, rotated, and averaged.

Preliminary Raw projection images show clear trimeric association anddistinct features of MOMP (FIG. 23D). In particular, in the illustrationof FIG. 23D, raw projection images of MOMP trimer show distinctstructural features (white arrow). The diameter of the MOMP trimer wascalculated to be 90 Angstroms.

Furthermore, preliminary 3D density maps were generated for comparisonto MOMP from Campylobacter jejuni (Protein Data Base (PDB) ID: 5LDT)[27](FIG. 23E). Class averaged images can be used for comparison topreviously solved MOMP structures, such as MOMP from Campylobacterjejuni (5LDT). Black arrow indicates the fitted edges of preliminarydensity map generated from class averages.

Example 2. Cell-Free Co-Translation Supports Soluble mMOMP Expression

Codon optimization was used to alter sequences for mMOMP-tNLP expressionin E. coli cell-free lysates (FIG. 2). Codon optimization of theΔ49ApoA1 and mMOMP sequences resulted in a ˜20% change in the primaryprotein coding sequences for both proteins (FIGS. 3A-3B, 12C, 16 and17). Co-translation reaction conditions using plasmids encoding Δ49ApoA1and mMOMP were initially screened using a bodipy-lysine fluorescentamino acid to simplify visualization of protein expression andsolubility screening.

The bodipy-lysine fluorescent amino acid is randomly inserted at lysinepositions within the protein at a low insertion rate. The mMOMP proteinis highly hydrophobic and is normally insoluble in the absence of anative lipid bilayer or detergents. Co-translation with both plasmids inthe presence of DMPC lipid alone did not result in a soluble mMOMPexpression product. Soluble mMOMP was observed only when the cell-freereactions were modified to include both DMPC lipid and telodendrimerPEG^(5k)-CA₈.

The solubility of mMOMP increased from 10% to 75% upon insertion intotNLP (FIGS. 4A-4B). The PEGylated tail of the telodendrimer may protectthe mMOMP from interacting with surrounding mMOMP-tNLPs and thusincreases its solubility.

After the cell-free reaction was completed, the total cell-free mixtureswere centrifuged by a table centrifuge at max speed for 10 minutes.After centrifugation, the supernatant was collected. MOMP solubility isdefined by the ratio of the amount MOMP protein in supernatant to theamount of MOMP protein in the total mixture.

In order to produce soluble mMOMP 0.1 to 0.5 mg/mL of telodendrimer wereprovided in the cell-free reaction.

By adding plasmids encoding mMOMP and scaffolding protein ApoA1 atdifferent ratio, the expressed ratios of mMOMP:ApoA1 and the number ofmMOMP per tNLP were controlled. Typically, the concentration of plasmidencoding mMOMP in the cell-free mixture is 15 ug/mL. Plasmid encodingApoA1 is added at a mMOMP plasmid to ApoA1 plasmid ratio of 1:1, 2:1,5:1, 10:1, 15:1, 20:1, 50:1, 100:1, or 200:1. The expressed ratios ofmMOMP:ApoA1 is assessed by SDS-PAGE. The optimal expressed ratios ofmMOMP:ApoA1 is expected to be from 1:1 to 3:1. The optimal expressedratios of mMOMP:ApoA1 is achieved by using mMOMP plasmid to ApoA1plasmid ratio from 10:1 to 25:1. At the optimal ration, the number ofmMOMP per tNLP is expected to be from 1 to 3 mMOMP per tNLP.

Reactions were scaled up to 1 mL to produce sufficient quantities ofmMOMP for subsequent nickel purification utilizing the HIS tag on theapolipoprotein scaffold component of the tNLP.

The purification provided a complex that was >95% pure based on SDS-PAGEanalysis. On average, a 1 mL reaction yielded 1.5 mg of purifiedmMOMP-tNLP (FIG. 5A) based on gel densitometry. Distinct bands indicatedthat the two proteins, apolipoprotein and mMOMP, were co-purifying as acomplex.

To further characterize the mMOMP-tNLP complex, individual affinitypurification elution fractions were assessed by size exclusionchromatography (SEC) (FIG. 5B). SEC analysis confirmed that eachmMOMP-tNLP fraction eluted at the appropriate time (retention time(t_(r))˜7 min) without un-incorporated protein or free lipid peaks(tr˜15 min and 4 min, respectively), indicating that the complex was ahomogenous mixture of mMOMP-tNLPs.

Dot blots of SEC fractions demonstrated that both the apolipoprotein andmMOMP were co-localized within the peak fraction (FIG. 5C).

Example 3. mMOMP-tNLPs Form Disc Shaped Nanoparticles

Dynamic light scattering (DLS) was used to visualize the overall size ofthe purified mMOMP-tNLP complex. The empty tNLPs were approximately 10nm in diameter (FIG. 6A). The mMOMP-tNLP particle sizes showed an almost4-fold increase in diameter to about 40 nm (FIG. 6B) wherein the termabout when referred to length indicates ±0.5 the unit of length such asnm or Å. This large increase was unexpected, but plausible given thateach mMOMP contains 16 transmembrane domains. In addition, imageanalysis using cryo-electron microscopy (cryoEM) indicated thatmMOMP-tNLPs were disc-shaped (FIG. 6C).

A comparison between empty tNLPs and mMOMP-tNLPs via cryoEM alsoconfirmed the larger particle size of mMOMP-tNLPs. The cryoEM imagesalso revealed that mMOMP-tNLPs, not empty tNLPs, contained multipleregions of enhanced density of relatively uniform size with a diameterof about 20-30 Å. Since the samples were highly purified, these regionslikely represent mMOMP proteins that form pores inside a tNLP.Interestingly, although the number of mMOMP pores per tNLP particlevaried, the mMOMP-tNLP particles had an average of 3 mMOMP proteinsinserted.

Example 4. mMOMP Associated with tNLPs Form Higher Order Structures

Membrane-bound porins are known to be resistant to denaturant, providinga means to probing the formation of oligomer species usingSDS-polyacrylamide gels [25, 28]. By analyzing mMOMP-tNLP in thepresence and absence of both heat and reducing agent, higher-orderoligomers of mMOMP were identified. SDS-PAGE of heated samples in thepresence of DTT showed primarily two distinct bands on the gel,corresponding to mMOMP and Δ49ApoA1 at approximately 40 kD and 22 kD,respectively. However, with heat and reducing agent (DTT) removed,distinct bands corresponding to mMOMP oligomers were observed on the gelthat were absent in tNLP alone control, indicating that these oligomersare part of mMOMP and not oligomers of the apolipoprotein scaffold (FIG.7A). These results closely resemble the gel banding pattern attributedto oligomer formation of native MOMP [28]. Western blot analysis probedwith mAb40 also indicated the formation of the higher order structuresof mMOMP (FIG. 7B). These multimeric structures are not evident inrecombinant MOMP produced in traditional E. coli expression systems,suggesting that the confinement to the constrained lipid bilayer of thetNLP can promote native-like oligomerization [25].

Dot blots were then tested to determine if adding both heat and reducingagent affect mMOMP antibody binding. Antibodies specific for mMOMPlinear epitope detection (mAb40) with and without heat and reducingagent resulted in the same intensity of signal, indicating that theoligomers of mMOMP are broken down to monomers upon heat and DTT.Furthermore, heat and DTT do not affect the mAb40 binding to mMOMP. As acontrol, mAb-HIS was always able to detect the apolipoprotein supportingscaffold (FIG. 8).

Native MOMP forms dimers, trimers, and tetramers in an oxidizedenvironment [15]. It has also been demonstrated that maintaining nativeMOMP structure is necessary to elicit a robust immune response [10, 19].The results in this Example show that mMOMP supported by tNLP particlesmimic native mMOMP oligomer structures. The mMOMP higher order oligomerresembles previously reported native MOMP trimers [19].

Example 5. tNLP Solubilized mMOMP Forms Functional Pores in Bilayers

Previous studies have shown that the presence of mMOMP initiates poresin lipid bilayers [15]. Therefore, we used conductance analysis to testthe function of mMOMP supported in the tNLP. The pore-forming activitiesof mMOMP-tNLP were tested in a typical black lipid membrane channelreconstitution experiment using the single-channel recording technique[29]. Control experiments with tNLP alone did not produce channelactivity under a series of applied transmembrane voltages ranging from−100 to +100 mV (FIG. 9A, trace i). However, discrete increases incurrent were observed 3-5 seconds after the addition of 1-2 μL ofmMOMP-tNLP solution to the cis-chamber. This current increasecorresponded to the bilayer pore formation by the mMOMP proteins,indicating functional mMOMP insertion (FIG. 9B, traces ii-iv). All mMOMPchannel incorporation events were permanent and did not show any gatingor transient blockade patterns under the conditions studied. ThemMOMP-tNLP conductance was predominantly ˜172 pA at physiologicalcondition.

The conductance change of a large number of incorporation events wasplotted on a histogram (n=184, FIG. 9B) and is consistent with thepresence of two gaussian peaks at 1× and 3× multiples of a singleconductance value. Interestingly, attempts to fit the histogram to a sumof three peaks at 1×, 2×, and 3× did not produce a better fit,indicating that mMOMP channel may have a tendency to oligomerize withinthe membrane and form trimers (corresponding to three pores).

Thus, the results of the conductance assays suggest that a population ofmMOMP in the mMOMP-tNLP sample is likely to be in a functionaloligomeric state in the bilayer. Accordingly, cell-free produced mMOMPappears to adopt a functional conformation, which has never beenpreviously reported for any recombinant MOMP. Importantly, cross-linkingof the recombinant protein was not required to observe oligomerization.Cell-free expression followed by direct insertion into the tNLP appearsto help maintain the functional conformation of membrane bound proteins[30, 31].

Example 6. The mMOMP-tNLP Complex Elicits an IgG Response in Mice

The tNLPs (negative control) or mMOMP-tNLPs were adjuvanted with CpG andinjected intramuscularly (i.m.) into mice. Additional groups of micewere injected i.m. with PBS (negative control) or Chlamydia EB (positivecontrol). It was found that mMOMP-tNLP supports the addition of CpGadjuvant and elicits significant levels of antigen-specific antibodytiters compared to CpG:tNLP (no antigen) and PBS controls (FIG. 10A).

The formulation of mMOMP-tNLP plus CpG adjuvant results in theincorporation of the CpG adjuvant into the mMOMP-tNLP particle.

Pooled mouse sera from injected mice were then probed on a western blotto detect for specific mMOMP binding (FIG. 10B). The sera from miceinjected with mMOMP-CpG-tNLP showed strong mMOMP binding. The lane fromsera immunized with Chlamydia EB also detected some mMOMP binding. It isnot surprising that EB sera showed less binding than mMOMP-CpG-tNLP serabecause EB contains many other proteins other than mMOMP. Therefore,there were many antibodies generated against EB and only a portion ofthese antibodies was mMOMP-specific. Sera from PBS and CpG:tNLP controlgroups showed no mMOMP binding. This Example shows that immunogenicadjuvants such as CpG can be incorporated into mMOMP-tNLP formulation.

Example 7: MOMP-NLP Complexes are Immunogenic and Protective

The protective response of MOMP-NLPs formulated with CpG, a TLR-9agonist that elicits Th1 responses, or CpG and FSL₁ was evaluated in amouse intranasal challenge study. (FIGS. 18A-18C).

FSL₁ is a TLR-2/6 agonist that induces Th2 response. It is expected thatwhen delivered together with antigens in the same NLP, the CpG and FSL₁will elicit more robust protective responses than if antigens andadjuvants were simply injected simultaneously. CpG and FSL₁ can beadministrated to the mouse using systemic and/or mucosal routes forimmunization.

Mice were inoculated intranasally with formulated controls or differentformulations of MOMP-NLPs with CpG or CpG and FSL₁ adjuvants. Withchlamydial challenges, the mice undergo weight loss and recovery. Therecovery is an indication of protection for any formulation.

In FIG. 18A, the antibody tiers from immunized mice show antibody crossreactivity with C. muridarum MOMP (anti-MOMP) or EB (anti-EB). In FIG.18B, weight loss over time following intranasal (i.n) challenge with C.muridarum was used as a measure of protection. Data was analyzed usingRM two-way ANOVA with Sidak's multiple comparison analysis.

Mice immunized with MOMP:CpG:NLPs or MOMP:CpG:FSL1:NLPs generatedantibodies that recognized both MOMP and EB (FIG. 18A). TheMOMP:CpG:FSL1:NLP formulation with two adjuvants showed a substantialprotective response in the intranasal model as compared to the otherformulations (FIG. 18B). Lower MOMP-specific IgG titers in the EB sampleis expected because there are other antigens, such as Pmps, presented onEB than just the MOMP protein. Additionally, mice immunized withMOMP:NLP lost significant body weight by 4 days post challenge (d.p.c.)but by 10 d.p.c. have recovered most of their weight (FIG. 18B).

FIG. 18C plots the number of Cm IFU recovered from mice vaccinated withdifferent MOMP:NLPs formulations. Each dot represents a mouse. Thehorizontal line corresponds to the median. The number of Cm IFUrecovered from mice vaccinated with MOMP:NLP was significantly less thanfrom sham-vaccinated groups (p<0.05).

These combined preliminary results demonstrate the feasibility ofextending NLP approach to the genital model for further vaccinedevelopment.

Additionally, since using systemic and/or mucosal routes forimmunization, a better protection has been observed when using bothroutes[32, 33]. It is therefore expected that delivery of CpG-1826 andFSL-1 by both routes will result in enhancing systemic and mucosalhumoral and cellular memory immune responses.

Example 8: Exemplary Chlamydia Vaccine Pipeline

NLPs provide a versatile platform for vaccine development. By combiningthe rapid production of functional membrane proteins with adjuvantaddition and structural screening, a pipeline for vaccine generation isdeveloped.

FIG. 19 illustrates a schematic of an exemplary Chlamydia vaccinepipeline. In particular, constituents such as DNA, lipids, cofactors andcell-free extracts are combined in a single reaction vial. The cell-freelysates utilize T7-coupled transcription and translation to producenanoparticles complexed with the antigens and adjuvants of interest. Thenanoparticles then can be characterized and administrated to mice toprotect them from a chlamydial infection.

Example 9: Generation of Optimal Vaccine Formulations

In this example, experiments were carrier out to optimize the ratio ofMOMP to Apolipoprotein for high-level cell-free expression,purification, and formulation of functional complexes in NLPs.

Cell-free expression technologies have demonstrated to overcomebottlenecks associated with membrane protein expression. In thisexample, cell-free C. muridarum MOMP have been generated in which theplasmid ratio of pApo to pMOMP was provided at 1:1, 1:5, 1:10, 1:25,1:50, and 1:100 as shown in FIG. 20A.

FIG. 20A shows results from the SDS-PAGE analysis following cell freesynthesis of MOMP-NLPs using varying amounts of fluorescent labeled Apoand MOMP proteins. The plasmid ratio expression screening displaysdifferent levels of inserted MOMP embedded in the NLPS complex. TheSDS-PAGE analysis results show that a higher ratio of MOMP to Apo leadsto a higher amount of MOMP proteins incorporated in the NLPs.

Scanning electron microscopy was also used to determine average particlesize of the MOMP-NLPs. FIG. 20B shows scanning electron microscopicimages (SEM) of (A) empty NLP disc, (B) MOMP-NLP disc with 1-2 monomersof MOMP inserted, (C) MOMP-NLP disc with 1-2 trimers of MOMP inserted,and (D) MOMP-NLP disc with >3 trimers of MOMP inserted.

The images of FIG. 20B demonstrate that the MOMP-NLP particles are disclike in shape and there are size differences among MOMP-NLP particleswith varying ratios of MOMP to Apo. The higher ratios of MOMP proteininserted in the disc correlate to particles with larger disc size.

Example 10: Cell-Free Production of Polymorphic Membrane ProteinsAssociated with MOMP

Polymorphic membrane proteins (Pmps) are another group of surfaceexposed candidate antigens. C. trachomatis and C. muridarum have ninePmp genes. Pmps are well conserved among all C. trachomatis serovars, aswell as C. muridarum. Therefore, Pmps may help broaden the protectiveimmune responses elicited by MOMP. This family of proteins is surfaceexposed and mediates the adhesion of Chlamydia EB to the eukaryotic hostcells. The Pmp proteins are also immunogenic in humans and mice.Vaccination of mice with fragments derived from different Pmps elicitsprotection against both genital and respiratory challenges with C.muridarum. Based on these studies, PmpC, PmpE, PmpF, PmpG and PmpH wereidentified as potential protective antigens [34-38]. However, productionof full-length recombinant Pmps has yet to be achieved.

In this example, experiments were carried out to engineer the expressionplasmids and produce water soluble full-length and truncated C.muridarum Pmp C, E, F, G, and H using cell-free approaches describedherein.

Exemplary PMP gene and protein sequences used in this study are listedin Table 3. The original sequences are retrieved from public databasessuch as Uniprot/SWISS-PROT or NCBI gene database as will be understoodby a person of ordinary skill in the art

TABLE 3 Exemplary full-length and truncated PMP C, E, F, G, and Hgene and protein sequences from C. muridarum SEQ Annotation SequencesID NO PmpCATGAAGTTTTTATCAGCTACCGCTGTATTTGCTGCAGCTCTTCCCTCTATCACAAGTGCTAGCTCCGTTGA20 TC_RS03520-ATCCCAAATAGAAACAAAAGATCTAAACTCTAGTAGAACAGGATCCTCATCATCGCAATCCTTCACTGAAA1246056 OriginalTAATTCCAGAAAATGGCGCAGAATATAGGGTATCTGGAGATGTTTCATTTTCTGATTTTTCAAATATACCAdatabaseGAAGAAGCAGAGACTCTTGCTATATCGCACAAAGAACAGCCTAATAACGAAGTAGTACTCTCCGAAGAAAAsequenceCCACCAAGCATCCTTTCAAGATTCTGCACAAAACCAAACTGAAAATGCCTCTGAAGGAAACTCTCCTAATAGCGAGAATACTAACCAGTCATCTACCACAGAAACCGAGTCTATAACTACTGATGAACAAGTGCAGAATGATAATGAATCTGCAGCTTCTGTACCTACTACTGTAGAAACAGCAACAGCTATGCGCCTCCCCTCTTACCATCTACAAACAGAATCATTAGTAGAAGGGGCTACAGAAGAAGATCAAAATCAACCGAACTCTCAAAATACATCTAGTGGCGGCGGAGCATTTTATAACTCTCAACAAGGACCTTTATCCTTTATCAATGATCCCGATAAAGACAGTTCTCTCACCTTATCAAAAATTCGAGTAATAGGAGAGGGTGGTGCCATTTACTCGAAAGGACCATTAAGCATAACAGGTCTTAAAAAATTAGCTTTAAAAGAAAACTTATCCCAAAAGGCTGGAGGAGCTATTTGTGCAGAATCCACTATTTCAATAAGTAGTGTAGATTCTATCATTTTTTCTAAGAATACAGTCACTCCTCCAGCTGCCAATAAACCTGAACTCCCTAACGATCCCTCTGGGAGTAATGGTAATGATGGTTCTGATGACAGTAACTCCTCAGGTAATACTGACTCAAATGAAAGCAACCCTAACAACAGCGCTTCTAATAACACTGGCTCTGAAAATGAGCTTTCTTCCAGTACCCCATCCGCACAACTTCCCAATCCCGCAACACCATTTTTATCATCTGTTTCTACAAACTCTCAACCTATAGACACAGAACCAGAAAATGCATGGCATGCTGAATCAGGGTCTGGAGGAGCTATCTATTCTAAAGGCAAGCTTTCTATCGCAAGCTCTAAAGAAGTAGTCTTCGATCACAACTCGGCCACCAAAAATGGAGGAGCTATCTTCGGAGAGGAAGAAATTGCTCTCGAAAAAATAGCGTCTCTGAAATTCGATTCCAACACTACCGGTGAAAAAGGTGGGGCTATTCATGCGAAAACAGTTACACTATCTGATATCAAAAACACTTTGATTTTCGTTAATAATACGGCTAAAACACCGGAAGAAAACTCTCTAAAATCTTCTCAACTAAACAACCAAAATCCTTCCGAAGAAGAGCACCAAGATACTAGTGAGGGTGAAGAAAGCCAGTCTCTTGAAACGTCACCTATAACTAATCAAGACTCTGCATCCTCTCATGTAGCCATTTTCCGTTCTATAGCAGCATCCTCCTCTCAATCTAATAGCGAAAATATCCCTAATGCAGATGGGTCTACATCTGCTGGGGGAGACGCAGGAAGCTCTTCACAACCATCGACACCAGGATCCGATTCTTCGATAAATCATGTGATTGGAGGAGGAGCTATCTATGGAGAGGCAGTCAAAATCGAGAACCTCTCTGGATATGGAACATTCTCCAACAATAACGCTGTTGATCATCAAATTTCTGGATCTACATCCGATGTTTTAGGAGGAGCTATCTATGCTAAAACATCACTAACTATCGATAGCGGGAACTCTAGTGGAACCATTACATTCTCTGAAAATACCACTTCTTCCAAATCTACAACAGGACAGGTTGCTGGAGGAGCCATCTTCTCCCCTAGTGTAACCATCACCACACCAGTGACCTTTTCTAAAAACTCTGCGATAAATGCCACAACCAGTTCTAAAAAGGATACCTTTGGGGGAGCTATCGGTGCAATCTCTACAGTTTCTCTATCCAAAGGAGCTCGATTCTCAGAAAATATTGCCGATCTTGGATCTGCTATTGGATTAGTACCTACTACACAAGATGCAGAAACTGTTCAGCTAACAACAGGTTCTTACTATTTTGAAAAGAATAAAGCACTAAAACGAGCAACTGTTTACGCTCCTATCGTATCTATCAAAGCTCATACCGCAACATTCGATCAAAATATCTCTGCAGAAGAAGGAAGCGCGATTTATTTCACTAAAGAAGCCACCATTGAGTCTTTGGGATCCGTTCTTTTTACAGGGAACTTGGTAACCCCAATACAAAGCACAACAGTGTTAACTTCTGGAAACACCTCAAAATACGGGGCTGCTATTTTTGGACAAATAGCGAATGCAAGCGGATCTCAAACTGATAACCTCCCCCTCAAACTGATCGCTTCTGGAGGGAATATCAGCTTCCGAAATAACGAATACCGTCCAGATGCCACTAATACTGGACAATCTACTTTCTGTAGTATCGCTGGAGATATTAAATTAACCATGCAGGCTGCAGAAGGCAAAGTAATCAGTTTCTTTGATGCTATACGAACTTCCACTAAGAAAACAGGAACTCTGGCCTCTGCTTATGACACACTAGATATCAATAAATCGAATGATTCAGGGTCCATAAATTCAGCCTTTACAGGGACCATTATGTTCTCCTCTGAATTACATGAGAACAAATCCTATATTCCACAAAACGTAGTCTTACACAGTGGCTCTCTCATATTGAAAGCAAATACGGAACTTCATGTGCTTTCGTTTGATCAGAAAGAAGGCTCTTCTCTTATTATGGAACCTGGATCTGTTCTTTCAAATCAAGATATTGCTGATGGTTCTTTAGTAGTAAATAGTCTTACCATTGATTTATCGAGTGTTGGAAGAAACAGTGCCTCTGGAGACAATATCTTCATGCCTCCAGAATTAAGAATCGTAGATACCTCTACAAATTCTGGAAACAGCTCTTCTACCCCGCCCTCATCGAATACACCACCAAACTCAACTCCGACAGCACAAGCTCCTATTTCCAAAAATTTTGCTGCCACAACCACGACACCAACAACACCTCCGACAACAGGGAACATCGTTTTCCTTAACGGAGTTATTAAACTGATTGATCCGAATGGGACATTTTTCCAAAACCCTGCATTAGGATCTGACCAAAAAATCTCTCTACTAGTACTCCCTTCAGATCAAACAAAACTCCAAGCTCAGAAAGTTGTGCTAACAGGAGACATCTCTCCTAAGAAAGGATACACAGGAACATTAACTCTTGATCCTCAACAATTACAAAATGGAGTAATCCAAGCTTTATGGACATTCAAATCCTACAGACAGTGGGCCTATATTCCTAGGGATAATCACTTTTATGCCAACTCGATTCTGGGATCCCAAATGTCTATGGCTACTGTCAAACAAGGATTAATCAATGATAAATTGAATCTTGCTCGCTTTGATGAGGTTGCTTACAATAATTTGTGGATATCAGGACTAGGAACCATGCTCTCTCAAAGAGGAGGCCAGCGATCAGAGGAAATGACTTATTACAGTAGAGGAGCTTCTGTTGCTTTAGATGCGAAACCTACCCAAGATTTGATCATTGGAGCAGCATTTAGTAAAATGATCGGAAGAAGCAAATCTTTGAAACTAGAGCGTAACTACACCCACAAGGGATCGGAATATTCCTACCAAGCATCGGTTTATGGAGGTAGTCCTTTCTATCTTACAATTAACAAAGAAGCAGGCCGATCCCTCCCTCTCTTATTACAAGGGGTTATCTCCTACGGATACATCAAACACGATACAGTTACCCACTATCCTACAATTCGTGAATTAAACAAAGGAGAGTGGGAAGACTTAGGATGGTTGACCGCTCTTCGAGTCTCTTCCATCTTAAAAACACCTAAACAAGGAGACTCCAAACGCATTACTGTTTACGGAGAAGTTGAATATTCTAGCATCCGTCAAAAACAATTTACGGAAACGGAATATGATCCTCGTTACTTCAGTAACTGCACCTATAGAAACTTAGCAGTTCCTGTAGGATTAGCCTTAGAGGGAGAATTCAAAGGTAACGATATTTTGATGTACAACAGATTCTCTGTAGCTTACATGCCATCCATCTATCGAAACTCTCCAGTATGCAAGTACCAAGTACTCTCATCTGGAGAAGGTGGAGAAATCGTCTGTGGTGTTCCCACCAGAAACTCCTCTCGAGCAGAATATAGTACGCAGTTATACCTTGGTCCTCTATGGACTTTATATGGATCCTACACATTAGAAGCGGACGCTCACACGTTAGCCAATATGATTAACTGTGGGGCTCGCATGACATTCTAA PmpCMKFLSATAVFAAALPSITSASSVESQIETKDLNSSRTGSSSSQSFTEIIPENGAEYRVSGDVSFSDFSNIP21 PMPC_CHLMUEEAETLAISHKEQPNNEVVLSEENHQASFQDSAQNQTENASEGNSPNSENTNQSSTTETESITTDEQVQNDNESAASVPTTVETATAMRLPSYHLQTESLVEGATEEDQNQPNSQNTSSGGGAFYNSQQGPLSFINDPDKDSSLTLSKIRVIGEGGAIYSKGPLSITGLKKLALKENLSQKAGGAICAESTISISSVDSIIFSKNTVTPPAANKPELPNDPSGSNGNDGSDDSNSSGNTDSNESNPNNSASNNTGSENELSSSTPSAQLPNPATPFLSSVSTNSQPIDTEPENAWHAESGSGGAIYSKGKLSIASSKEVVFDHNSATKNGGAIFGEEEIALEKIASLKFDSNTTGEKGGAIHAKTVTLSDIKNTLIFVNNTAKTPEENSLKSSQLNNQNPSEEEHQDTSEGEESQSLETSPITNQDSASSHVAIFRSIAASSSQSNSENIPNADGSTSAGGDAGSSSQPSTPGSDSSINHVIGGGAIYGEAVKIENLSGYGTFSNNNAVDHQISGSTSDVLGGAIYAKTSLTIDSGNSSGTITFSENTTSSKSTTGQVAGGAIFSPSVTITTPVTFSKNSAINATTSSKKDTFGGAIGAISTVSLSKGARFSENIADLGSAIGLVPTTQDAETVQLTTGSYYFEKNKALKRATVYAPIVSIKAHTATFDQNISAEEGSAIYFTKEATIESLGSVLFTGNLVTPIQSTTVLTSGNTSKYGAAIFGQIANASGSQTDNLPLKLIASGGNISFRNNEYRPDATNTGQSTFCSIAGDIKLTMQAAEGKVISFFDAIRTSTKKTGTLASAYDTLDINKSNDSGSINSAFTGTIMESSELHENKSYIPQNVVLHSGSLILKANTELHVLSFDQKEGSSLIMEPGSVLSNQDIADGSLVVNSLTIDLSSVGRNSASGDNIFMPPELRIVDTSTNSGNSSSTPPSSNTPPNSTPTAQAPISKNFAATTTTPTTPPTTGNIVFLNGVIKLIDPNGTFFQNPALGSDQKISLLVLPSDQTKLQAQKVVLTGDISPKKGYTGTLTLDPQQLQNGVIQALWTFKSYRQWAYIPRDNHFYANSILGSQMSMATVKQGLINDKLNLARFDEVAYNNLWISGLGTMLSQRGGQRSEEMTYYSRGASVALDAKPTQDLIIGAAFSKMIGRSKSLKLERNYTHKGSEYSYQASVYGGSPFYLTINKEAGRSLPLLLQGVISYGYIKHDTVTHYPTIRELNKGEWEDLGWLTALRVSSILKTPKQGDSKRITVYGEVEYSSIRQKQFTETEYDPRYFSNCTYRNLAVPVGLALEGEFKGNDILMYNRFSVAYMPSIYRNSPVCKYQVLSSGEGGEIVCGVPTRNSSRAEYSTQLYLGPLWTLYGSYTLEADAHTLANMINCGARMTF PmpCCATATGAGCAGCGTTGAATCCCAAATAGAAACAAAAGATCTGAACTCTAGTCGCACAGGCTCCTCATCATC22 PmpC_EcOpt_dSig:GCAATCCTTCACTGAAATAATTCCAGAAAATGGCGCAGAATATCGCGTATCTGGAGATGTTTCATTTTCTGE. coli codonATTTTTCAAATATACCAGAAGAAGCAGAGACTCTTGCTATATCGCACAAAGAACAGCCTAATAACGAAGTAoptimized,GTACTCTCCGAAGAAAACCACCAAGCATCCTTTCAAGATTCTGCACAAAACCAAACTGAAAATGCCTCTGAN-terminalAGGAAACTCTCCTAATAGCGAGAATACTAACCAGTCATCTACCACAGAAACCGAGTCTATAACTACTGATGnuclearAACAAGTGCAGAATGATAATGAATCTGCAGCTTCTGTACCTACTACTGTAGAAACAGCAACAGCTATGCGClocalizationCTCCCCTCTTACCATCTGCAAACAGAATCATTAGTAGAAGGGGCTACAGAAGAAGATCAAAATCAACCGAAsignal removedCTCTCAAAATACATCTAGTGGCGGCGGAGCATTTTATAACTCTCAACAAGGACCTTTATCCTTTATCAATGATCCCGATAAAGACAGTTCTCTCACCTTATCAAAAATTCGAGTAATAGGAGAGGGTGGTGCCATTTACTCGAAAGGACCATTAAGCATAACAGGTCTTAAAAAATTAGCTTTAAAAGAAAACTTATCCCAAAAGGCTGGAGGAGCTATTTGTGCAGAATCCACTATTTCAATAAGTAGTGTAGATTCTATCATTTTTTCTAAGAATACAGTCACTCCTCCAGCTGCCAATAAACCTGAACTCCCTAACGATCCCTCTGGGAGTAATGGTAATGATGGTTCTGATGACAGTAACTCCTCAGGTAATACTGACTCAAATGAAAGCAACCCTAACAACAGCGCTTCTAATAACACTGGCTCTGAAAATGAGCTTTCTTCCAGTACCCCATCCGCACAACTTCCCAATCCCGCAACACCATTTTTATCATCTGTTTCTACAAACTCTCAACCTATAGACACAGAACCAGAAAATGCATGGCATGCTGAATCAGGGTCTGGAGGAGCTATCTATTCTAAAGGCAAACTTTCTATCGCAAGCTCTAAAGAAGTAGTCTTCGATCACAACTCGGCCACCAAAAATGGAGGAGCTATCTTCGGAGAGGAAGAAATTGCTCTCGAAAAAATAGCGTCTCTGAAATTCGATTCCAACACTACCGGTGAAAAAGGTGGGGCTATTCATGCGAAAACAGTTACACTGTCTGACATCAAAAACACTTTGATTTTCGTTAATAATACGGCTAAAACACCGGAAGAAAACTCTCTGAAATCTTCTCAACTGAACAACCAAAATCCTTCCGAAGAAGAGCACCAAGATACTAGTGAGGGTGAAGAAAGCCAGTCTCTTGAAACGTCACCTATAACTAATCAAGACTCTGCATCCTCTCATGTAGCCATTTTCCGTTCTATAGCAGCATCCTCCTCTCAATCTAATAGCGAAAATATCCCTAATGCAGATGGGTCTACATCTGCTGGGGGAGACGCAGGAAGCTCTTCACAACCATCGACACCAGGCTCCGATTCTTCGATAAATCATGTGATTGGAGGAGGAGCTATCTATGGAGAGGCAGTCAAAATCGAGAACCTCTCTGGATATGGAACATTCTCCAACAATAACGCTGTTGATCATCAAATTTCTGGATCTACATCCGATGTTTTAGGAGGAGCTATCTATGCTAAAACATCACTGACTATCGATAGCGGGAACTCTAGTGGAACCATTACATTCTCTGAAAATACCACTTCTTCCAAATCTACAACAGGACAGGTTGCTGGAGGAGCCATCTTCTCCCCTAGTGTAACCATCACCACACCAGTGACCTTTTCTAAAAACTCTGCGATAAATGCCACAACCAGTTCTAAAAAGGATACCTTTGGGGGAGCTATCGGTGCAATCTCTACAGTTTCTCTGTCCAAAGGAGCCCGATTCTCAGAAAATATTGCCGATCTTGGATCTGCTATTGGATTAGTACCTACTACACAAGATGCAGAAACTGTTCAGCTGACAACAGGTTCTTACTATTTTGAAAAGAATAAAGCACTGAAACGAGCAACTGTTTACGCTCCTATCGTATCTATCAAAGCTCATACCGCAACATTCGATCAAAATATCTCTGCAGAAGAAGGAAGCGCGATTTATTTCACTAAAGAAGCCACCATTGAGTCTTTGGGTTCCGTTCTTTTTACAGGGAACTTGGTAACCCCAATACAAAGCACAACAGTGTTAACTTCTGGAAACACCTCAAAATACGGGGCTGCTATTTTTGGACAAATAGCGAATGCAAGCGGATCTCAAACTGATAACCTCCCCCTCAAACTGATCGCTTCTGGAGGGAATATCAGCTTCCGAAATAACGAATACCGTCCAGATGCCACTAATACTGGACAATCTACTTTCTGTAGTATCGCTGGAGATATTAAATTAACCATGCAGGCTGCAGAAGGCAAAGTAATCAGTTTCTTTGATGCTATACGAACTTCCACTAAGAAAACAGGAACTCTGGCCTCTGCTTATGACACACTGGATATCAATAAATCGAATGATTCAGGGTCCATAAATTCAGCCTTTACAGGGACCATTATGTTCTCCTCTGAGCTCCATGAGAACAAATCCTATATTCCACAAAACGTAGTCTTACACAGTGGCTCTCTCATATTGAAAGCAAATACGGAACTTCATGTGCTTTCGTTTGATCAGAAAGAAGGCTCTTCTCTTATTATGGAACCTGGATCTGTTCTTTCAAATCAAGATATTGCTGATGGTTCTTTAGTAGTAAATAGTCTTACCATTGATTTATCGAGTGTTGGACGCAACAGTGCCTCTGGAGACAATATCTTCATGCCTCCAGAATTACGCATCGTAGATACCTCTACAAATTCTGGAAACAGCTCTTCTACCCCGCCCTCATCGAATACACCACCAAACTCAACTCCGACAGCACAAGCTCCTATTTCCAAAAATTTTGCTGCCACAACCACGACACCAACAACACCTCCGACAACAGGGAACATCGTTTTCCTTAACGGAGTTATTAAACTGATTGATCCGAATGGGACATTTTTCCAAAACCCTGCATTAGGATCTGACCAAAAAATCTCTCTGCTGGTACTCCCTTCAGATCAAACAAAACTCCAAGCTCAGAAAGTTGTGCTGACAGGAGACATCTCTCCTAAGAAAGGATACACAGGAACATTAACTCTTGATCCTCAACAATTACAAAATGGAGTAATCCAAGCCTTATGGACATTCAAATCCTACCGCCAGTGGGCCTATATTCCTCGCGATAATCACTTTTATGCCAACTCGATTCTGGGTTCCCAAATGTCCATGGCTACTGTCAAACAAGGATTAATCAATGATAAATTGAATCTTGCTCGCTTTGATGAGGTTGCTTACAATAATTTGTGGATATCAGGACTGGGAACCATGCTCTCTCAACGCGGAGGCCAGCGATCAGAGGAAATGACTTATTACAGTCGCGGAGCTTCTGTTGCTTTAGATGCGAAACCTACCCAAGATTTGATCATTGGAGCAGCATTTAGTAAAATGATCGGACGCAGCAAATCTTTGAAACTGGAGCGTAACTACACCCACAAGGGATCGGAATATTCCTACCAAGCATCGGTTTATGGAGGTAGTCCTTTCTATCTTACAATTAACAAAGAAGCAGGCCGATCCCTCCCTCTCTTATTACAAGGGGTTATCTCCTACGGATACATCAAACACGATACAGTTACCCACTATCCTACAATTCGTGAATTAAACAAAGGAGAGTGGGAAGACTTAGGATGGTTGACCGCTCTTCGAGTCTCTTCCATCTTAAAAACACCTAAACAAGGAGACTCCAAACGCATTACTGTTTACGGAGAAGTTGAATATTCTAGCATCCGTCAAAAACAATTTACGGAAACGGAATATGATCCTCGTTACTTCAGTAACTGCACCTATCGCAACTTAGCAGTTCCTGTAGGATTAGCCTTAGAGGGAGAATTCAAAGGTAACGATATTTTGATGTACAACCGCTTCTCTGTAGCTTACATGCCATCCATCTATCGAAACTCTCCAGTATGCAAGTACCAAGTACTCTCATCTGGAGAAGGTGGAGAAATCGTCTGTGGTGTTCCCACCCGCAACTCCTCCCGAGCAGAATATAGTACGCAGTTATACCTTGGTCCTCTGTGGACTTTATATGGCTCCTACACATTAGAAGCGGACGCTCACACGTTAGCCAATATGATTAACTGTGGGGCTCGCATGACATTCtaaGGATCC PmpCMSSVESQIETKDLNSSRTGSSSSQSFTEIIPENGAEYRVSGDVSFSDFSNIPEEAETLAISHKEQPNNEVV23 PmpC_EcOpt_dSig:LSEENHQASFQDSAQNQTENASEGNSPNSENTNQSSTTETESITTDEQVQNDNESAASVPTTVETATAMRLE. coli codonPSYHLQTESLVEGATEEDQNQPNSQNTSSGGGAFYNSQQGPLSFINDPDKDSSLTLSKIRVIGEGGAIYSKoptimized,GPLSITGLKKLALKENLSQKAGGAICAESTISISSVDSIIFSKNTVTPPAANKPELPNDPSGSNGNDGSDDN-terminalSNSSGNTDSNESNPNNSASNNTGSENELSSSTPSAQLPNPATPFLSSVSTNSQPIDTEPENAWHAESGSGGnuclearAIYSKGKLSIASSKEVVEDHNSATKNGGAIFGEEEIALEKIASLKFDSNTTGEKGGAIHAKTVTLSDIKNTlocalizationLIFVNNTAKTPEENSLKSSQLNNQNPSEEEHQDTSEGEESQSLETSPITNQDSASSHVAIFRSIAASSSQSsignal removedNSENIPNADGSTSAGGDAGSSSQPSTPGSDSSINHVIGGGAIYGEAVKIENLSGYGTFSNNNAVDHQISGSTSDVLGGAIYAKTSLTIDSGNSSGTITFSENTTSSKSTTGQVAGGAIFSPSVTITTPVTFSKNSAINATTSSKKDTEGGAIGAISTVSLSKGARFSENIADLGSAIGLVPTTQDAETVQLTTGSYYFEKNKALKRATVYAPIVSIKAHTATFDQNISAEEGSAIYFTKEATIESLGSVLFTGNLVTPIQSTTVLTSGNTSKYGAAIFGQTANASGSQTDNLPLKLIASGGNISFRNNEYRPDATNTGQSTFCSIAGDIKLTMQAAEGKVISFFDAIRTSTKKTGTLASAYDTLDINKSNDSGSINSAFTGTIMESSELHENKSYIPQNVVLHSGSLILKANTELHVLSFDQKEGSSLIMEPGSVLSNQDIADGSLVVNSLTIDLSSVGRNSASGDNIFMPPELRIVDTSTNSGNSSSTPPSSNTPPNSTPTAQAPISKNFAATTTTPTTPPTTGNIVFLNGVIKLIDPNGTFFQNPALGSDQKISLLVLPSDQTKLQAQKVVLTGDISPKKGYTGTLTLDPQQLQNGVIQALWTFKSYRQWAYIPRDNHFYANSILGSQMSMATVKQGLINDKLNLARFDEVAYNNLWISGLGTMLSQRGGQRSEEMTYYSRGASVALDAKPTQDLIIGAAFSKMIGRSKSLKLERNYTHKGSEYSYQASVYGGSPFYLTINKEAGRSLPLLLQGVISYGYIKHDTVTHYPTIRELNKGEWEDLGWLTALRVSSILKTPKQGDSKRITVYGEVEYSSIRQKQFTETEYDPRYFSNCTYRNLAVPVGLALEGEFKGNDILMYNRFSVAYMPSIYRNSPVCKYQVLSSGEGGEIVCGVPTRNSSRAEYSTQLYLGPLWTLYGSYTLEADAHTLANMINCGARMTF PmpCCATATGAGCAGCGTTGAATCCCAAATAGAAACAAAAGATCTGAACTCTAGTCGCACAGGCTCCTCATCATC24 PmpC_EcOpt_dSig_GCAATCCTTCACTGAAATAATTCCAGAAAATGGCGCAGAATATCGCGTATCTGGAGATGTTTCATTTTCTGdPMPATTTTTCAAATATACCAGAAGAAGCAGAGACTCTTGCTATATCGCACAAAGAACAGCCTAATAACGAAGTAE. coli codonGTACTCTCCGAAGAAAACCACCAAGCATCCTTTCAAGATTCTGCACAAAACCAAACTGAAAATGCCTCTGAoptimized,AGGAAACTCTCCTAATAGCGAGAATACTAACCAGTCATCTACCACAGAAACCGAGTCTATAACTACTGATGN-terminalAACAAGTGCAGAATGATAATGAATCTGCAGCTTCTGTACCTACTACTGTAGAAACAGCAACAGCTATGCGCnuclearCTCCCCTCTTACCATCTGCAAACAGAATCATTAGTAGAAGGGGCTACAGAAGAAGATCAAAATCAACCGAAlocalizationCTCTCAAAATACATCTAGTGGCGGCGGAGCATTTTATAACTCTCAACAAGGACCTTTATCCTTTATCAATGsignal removed,ATCCCGATAAAGACAGTTCTCTCACCTTATCAAAAATTCGAGTAATAGGAGAGGGTGGTGCCATTTACTCGadhesion domainAAAGGACCATTAAGCATAACAGGTCTTAAAAAATTAGCTTTAAAAGAAAACTTATCCCAAAAGGCTGGAGG(ChlamPMP_M)AGCTATTTGTGCAGAATCCACTATTTCAATAAGTAGTGTAGATTCTATCATTTTTTCTAAGAATACAGTCAremovedCTCCTCCAGCTGCCAATAAACCTGAACTCCCTAACGATCCCTCTGGGAGTAATGGTAATGATGGTTCTGATGACAGTAACTCCTCAGGTAATACTGACTCAAATGAAAGCAACCCTAACAACAGCGCTTCTAATAACACTGGCTCTGAAAATGAGCTTTCTTCCAGTACCCCATCCGCACAACTTCCCAATCCCGCAACACCATTTTTATCATCTGTTTCTACAAACTCTCAACCTATAGACACAGAACCAGAAAATGCATGGCATGCTGAATCAGGGTCTGGAGGAGCTATCTATTCTAAAGGCAAACTTTCTATCGCAAGCTCTAAAGAAGTAGTCTTCGATCACAACTCGGCCACCAAAAATGGAGGAGCTATCTTCGGAGAGGAAGAAATTGCTCTCGAAAAAATAGCGTCTCTGAAATTCGATTCCAACACTACCGGTGAAAAAGGTGGGGCTATTCATGCGAAAACAGTTACACTGTCTGACATCAAAAACACTTTGATTTTCGTTAATAATACGGCTAAAACACCGGAAGAAAACTCTCTGAAATCTTCTCAACTGAACAACCAAAATCCTTCCGAAGAAGAGCACCAAGATACTAGTGAGGGTGAAGAAAGCCAGTCTCTTGAAACGTCACCTATAACTAATCAAGACTCTGCATCCTCTCATGTAGCCATTTTCCGTTCTATAGCAGCATCCTCCTCTCAATCTAATAGCGAAAATATCCCTAATGCAGATGGGTCTACATCTGCTGGGGGAGACGCAGGAAGCTCTTCACAACCATCGACACCAGGCTCCGATTCTTCGATAAATCATGTGATTGGAGGAGGAGCTATCTATGGAGAGGCAGTCAAAATCGAGAACCTCTCTGGATATGGAACATTCTCCAACAATAACGCTGTTGATCATCAAATTTCTGGATCTACATCCGATGTTTTAGGAGGAGCTATCTATGCTAAAACATCACTGACTATCGATAGCGGGAACTCTAGTGGAACCATTACATTCTCTGAAAATACCACTTCTTCCAAATCTACAACAGGACAGGTTGCTGGAGGAGCCATCTTCTCCCCTAGTGTAACCATCACCACACCAGTGACCTTTTCTAAAAACTCTGCGATAAATGCCACAACCAGTTCTAAAAAGGATACCTTTGGGGGAGCTATCGGTGCAATCTCTACAGTTTCTCTGTCCAAAGGAGCCCGATTCTCAGAAAATATTGCCGATCTTGGATCTGCTATTGGATTAGTACCTACTACACAAGATGCAGAAACTGTTCAGCTGACAACAGGTTCTTACTATTTTGAAAAGAATAAAGCACTGAAACGAGCAACTGTTTACGCTCCTATCGTATCTATCAAAGCTCATACCGCAACATTCGATCAAAATATCTCTGCAGAAGAAGGAAGCGCGATTTATTTCACTAAAGAAGCCACCATTGAGTCTTTGGGTTCCGTTCTTTTTACAGGGAACTTGGTAACCCCAATACAAAGCACAACAGTGTTAACTTCTGGAAACACCTCAAAATACGGGGCTGCTATTTTTGGACAAATAGCGAATGCAAGCGGATCTCAAACTGATAACCTCCCCCTCAAACTGATCGCTTCTGGAGGGAATATCAGCTTCCGAAATAACGAATACCGTCCAGATGCCACTAATACTGGACAATCTACTTTCTGTAGTATCGCTGGAGATATTAAATTAACCATGCAGGCTGCAGAAGGCAAAGTAATCAGTTTCTTTGATGCTATACGAACTTCCACTAAGAAAACAGGAACTCTGGCCTCTGCTTATGACACACTGGATATCAATAAATCGAATGATTCAGGGTCCATAAATTCAGCCTTTACAGGGACCATTATGTTCTCCTCTGAGCTCCATGAGAACAAATCCTATATTCCACAAAACGTAGTCTTACACAGTAAATCCTACCGCCAGTGGGCCTATATTCCTCGCGATAATCACTTTTATGCCAACTCGATTCTGGGTTCCCAAATGTCCATGGCTACTGTCAAACAAGGATTAATCAATGATAAATTGAATCTTGCTCGCTTTGATGAGGTTGCTTACAATAATTTGTGGATATCAGGACTGGGAACCATGCTCTCTCAACGCGGAGGCCAGCGATCAGAGGAAATGACTTATTACAGTCGCGGAGCTTCTGTTGCTTTAGATGCGAAACCTACCCAAGATTTGATCATTGGAGCAGCATTTAGTAAAATGATCGGACGCAGCAAATCTTTGAAACTGGAGCGTAACTACACCCACAAGGGATCGGAATATTCCTACCAAGCATCGGTTTATGGAGGTAGTCCTTTCTATCTTACAATTAACAAAGAAGCAGGCCGATCCCTCCCTCTCTTATTACAAGGGGTTATCTCCTACGGATACATCAAACACGATACAGTTACCCACTATCCTACAATTCGTGAATTAAACAAAGGAGAGTGGGAAGACTTAGGATGGTTGACCGCTCTTCGAGTCTCTTCCATCTTAAAAACACCTAAACAAGGAGACTCCAAACGCATTACTGTTTACGGAGAAGTTGAATATTCTAGCATCCGTCAAAAACAATTTACGGAAACGGAATATGATCCTCGTTACTTCAGTAACTGCACCTATCGCAACTTAGCAGTTCCTGTAGGATTAGCCTTAGAGGGAGAATTCAAAGGTAACGATATTTTGATGTACAACCGCTTCTCTGTAGCTTACATGCCATCCATCTATCGAAACTCTCCAGTATGCAAGTACCAAGTACTCTCATCTGGAGAAGGTGGAGAAATCGTCTGTGGTGTTCCCACCCGCAACTCCTCCCGAGCAGAATATAGTACGCAGTTATACCTTGGTCCTCTGTGGACTTTATATGGCTCCTACACATTAGAAGCGGACGCTCACACGTTAGCCAATATGATTAACTGTGGGGCTCGCATGACATTCtaaGGATCC PmpCMSSVESQIETKDLNSSRTGSSSSQSFTEIIPENGAEYRVSGDVSFSDFSNIPEEAETLAISHKEQPNNEVV25 PmpC_EcOpt_dSig_LSEENHQASFQDSAQNQTENASEGNSPNSENTNQSSTTETESITTDEQVQNDNESAASVPTTVETATAMRLdPMPPSYHLQTESLVEGATEEDQNQPNSQNTSSGGGAFYNSQQGPLSFINDPDKDSSLTLSKIRVIGEGGAIYSKE. coli codonGPLSITGLKKLALKENLSQKAGGAICAESTISISSVDSIIFSKNTVTPPAANKPELPNDPSGSNGNDGSDDoptimized,SNSSGNTDSNESNPNNSASNNTGSENELSSSTPSAQLPNPATPFLSSVSTNSQPIDTEPENAWHAESGSGGN-terminalAIYSKGKLSIASSKEVVEDHNSATKNGGAIFGEEEIALEKIASLKFDSNTTGEKGGAIHAKTVTLSDIKNTnuclearLIFVNNTAKTPEENSLKSSQLNNQNPSEEEHQDTSEGEESQSLETSPITNQDSASSHVAIFRSIAASSSQSlocalizationNSENIPNADGSTSAGGDAGSSSQPSTPGSDSSINHVIGGGAIYGEAVKIENLSGYGTFSNNNAVDHQISGSsignal removed,TSDVLGGAIYAKTSLTIDSGNSSGTITFSENTTSSKSTTGQVAGGAIFSPSVTITTPVTFSKNSAINATTSadhesion domainSKKDTEGGAIGAISTVSLSKGARFSENIADLGSAIGLVPTTQDAETVQLTTGSYYFEKNKALKRATVYAPI(ChlamPMP_M)VSIKAHTATFDQNISAEEGSAIYFTKEATIESLGSVLFTGNLVTPIQSTTVLTSGNTSKYGAAIFGQTANAremovedSGSQTDNLPLKLIASGGNISFRNNEYRPDATNTGQSTFCSIAGDIKLTMQAAEGKVISFFDAIRTSTKKTGTLASAYDTLDINKSNDSGSINSAFTGTIMESSELHENKSYIPQNVVLHSKSYRQWAYIPRDNHFYANSILGSQMSMATVKQGLINDKLNLARFDEVAYNNLWISGLGTMLSQRGGQRSEEMTYYSRGASVALDAKPTQDLIIGAAFSKMIGRSKSLKLERNYTHKGSEYSYQASVYGGSPFYLTINKEAGRSLPLLLQGVISYGYIKHDTVTHYPTIRELNKGEWEDLGWLTALRVSSILKTPKQGDSKRITVYGEVEYSSIRQKQFTETEYDPRYFSNCTYRNLAVPVGLALEGEFKGNDILMYNRFSVAYMPSIYRNSPVCKYQVLSSGEGGEIVCGVPTRNSSRAEYSTQLYLGPLWTLYGSYTLEADAHTLANMINCGARMTF PmpDATGAGTTCCGAGAAAGATAAAAAAAACTCCTGTTCTAAGTTTTCCTTATCGGTAGTAGCAGCTATTCTCGC26 TC_RS01000-TTCTATGAGTGGTTTATCGAATTGTTCCGATCTTTATGCCGTAGGAAGTTCTGCAGACCATCCTGCCTACT1246323 OriginalTGATTCCTCAAGCGGGGTTATTATTGGATCATATTAAGGATATATTCATTGGCCCTAAAGATAGTCAGGATdatabase sequenceAAGGGGCAGTATAAGTTGATTATTGGTGAGGCTGGCTCTTTCCAAGATAGTAATGCAGAGACTCTTCCTCAAAAGGTAGAGCACAGCACTTTGTTTTCAGTTACAACACCTATAATTGTGCAAGGAATAGATCAACAAGATCAGGTCTCTTCGCAGGGATTGGTCTGTAATTTTTCAGGAGATCATTCAGAGGAGATTTTTGAGAGAGAATCCTTTTTAGGGATCGCTTTCCTAGGGAATGGTAGCAAGGATGGAATCACGTTAACAGATATAAAATCTTCGTTATCTGGTGCTGCCTTGTATTCTTCAGATGATCTTATTTTTGAAAGAATTAAGGGAGATATAGAGCTTTCTTCTTGTTCATCTTTAGAAAGAGGAGGAGCTTGTTCAGCTCAAAGTATTTTAATTCATGATTGTCAAGGATTAACGGTAAAACATTGTGCCGCAGGGGTGAATGTTGAAGGAGTTAGTGCTAGCGACCATCTCGGATTTGGGGGCGGGGCCTTCTCTACTACAAGTTCTCTTTCTGGAGAGAAGAGTTTGTATATGCCTGCAGGCGATATTGTGGTGGCTACCTGCGATGGTCCTGTGTGTTTCGAAGGAAATAGTGCTCAGTTAGCAAATGGTGGCGCTATTGCCGCTTCTGGTAAAGTTCTTTTTGTAGCTAACGAAAAAAAGATTTCCTTTACAGACAACCAAGCTTTGTCTGGAGGAGCTATTTCTGCATCTTCTAGTATTTCTTTCCAAAATTGTGCTGAGCTTGTGTTCAAGAGTAATCTTGCAAAAGGAGTTAAAGATAAATGTTCTTTGGGAGGAGGTGCTTTAGCCTCTTTAGAATCCGTAGTTTTGAAAGATAATCTCGGTATTACTTATGAAAAAAATCAGTCCTATTCGGAAGGAGGGGCTATTTTTGGGAAGGATTGTGAGATTTTTGAAAACAGGGGGCCTGTTGTATTCAGAGATAATACAGCTGCTTTAGGAGGCGGAGCTATTTTGGCGCAACAAACTGTGGCGATTTGTGGTAATAAGTCTGGAATATCTTTTGAAGGAAGTAAGTCTAGTTTTGGAGGGGCCATTGCTTGTGGAAATTTCTCTTCTGAGAATAATTCTTCAGCTTTGGGATCAATTGATATCTCTAACAATCTAGGAGATATCTCTTTTCTTCGGACTCTGTGTACTACTTCGGATTTAGGGCAAACGGATTACCAAGGGGGAGGGGCCTTATTCGCTGAAAATATTTCTCTTTCTGAGAATGCTGGTGCAATTACTTTCAAAGACAATATTGTGAAGACATTTGCCTCAAATGGAAAAATGTTGGGTGGAGGGGCAATTTTAGCTTCAGGAAATGTTTTGATTAGCAAAAACTCTGGAGAGATTTCTTTTGTAGGGAATGCTCGAGCTCCTCAGGCTATTCCGACTCGTTCATCTGACGAATTGTCTTTTGGCGCACAATTAACTCAAACTACTTCAGGATGTTCTGGAGGAGGAGCTCTTTTTGGTAAAGAGGTTGCCATTGTTCAAAATGCCACTGTTGTATTCGAGCAAAATCGCTTACAGTGTGGCGAGCAGGAAACACATGGTGGAGGCGGTGCTGTTTATGGTATGGAGAGTGCCTCTATTATTGGAAACTCTTTTGTGAGATTCGGAAATAATTACGCTGTAGGGAATCAGATTTCTGGAGGAGCTCTTTTATCCAAGAAGGTCCGTTTAGCTGAAAATACAAGGGTAGATTTTTCTCGAAATATCGCTACTTTCTGCGGCGGGGCTGTTCAAGTTTCTGATGGAAGTTGCGAATTGATCAACAATGGGTATGTGCTATTCAGAGATAACCGAGGGCAGACATTTGGTGGGGCTATTTCTTGCTTGAAAGGAGATGTGATCATTTCCGGAAATAAAGATAGGGTTGAGTTTAGAGATAACATTGTGACGCGGCCTTATTTTGAAGAAAATGAAGAAAAAGTTGAGACAGCAGATATTAATTCAGATAAGCAAGAAGCAGAAGAGCGCTCTTTATTAGAGAACATTGAGCAGAGCTTTATTACTGCAACTAATCAGACCTTTTTCTTAGAGGAAGAGAAACTCCCATCAGAAGCTTTTATCTCTGCTGAAGAACTTTCAAAGAGAAGAGAATGTGCTGGTGGGGCGATTTTTGCAAAACGGGTCTACATTACGGATAATAAAGAACCTATCTTGTTTTCGCATAATTTTTCTGATGTTTATGGGGGAGCTATTTTTACGGGTTCTCTACAGGAAACTGATAAACAAGATGTTGTAACTCCTGAAGTTGTGATATCAGGCAACGATGGGGATGTCATTTTTTCTGGAAATGCAGCTAAACATGATAAGCATTTACCTGATACAGGTGGTGGAGCCATTTGTACACAGAATTTGACGATTTCCCAAAACAATGGGAATGTCTTGTTCTTGAACAATTTTGCTTGTTCTGGTGGAGCAGTTCGCATAGAGGATCATGGAGAAGTTCTTTTAGAGGCTTTTGGGGGAGATATTATTTTCAATGGAAACTCTTCTTTCAGAGCTCAAGGATCGGATGCGATCTATTTTGCTGGTAAGGACTCTAGAATTAAAGCTTTAAATGCTACTGAAGGACATGCGATTGTGTTCCAAGATGCATTGGTGTTTGAAAATATAGAAGAAAGAAAGTCTTCGGGACTATTGGTGATTAACTCTCAGGAAAATGAGGGTTATACGGGATCCGTCCGATTTTTAGGATCTGAAAGTAAGGTTCCTCAATGGATTCATGTGCAACAGGGAGGTCTTGAGTTGCTACATGGAGCTATTTTATGTAGTTATGGGGTTAAACAAGATCCTAGAGCTAAAATAGTATTATCTGCTGGATCTAAATTGAAGATTCTAGATTCAGAGCAAGAAAATAACGCAGAAATTGGAGATCTTGAAGATTCTGTTAATTCAGAAAAAACACCATCTCTTTGGATTGGGAAGAACGCTCAAGCAAAAGTCCCTCTGGTTGATATCCATACTATTTCTATTGATTTAGCATCATTTTCTTCTAAAGCTCAGGAAACCCCTGAGGAAGCTCCACAAGTCATCGTCCCTAAGGGAAGTTGTGTCCACTCGGGAGAGTTAAGTTTGGAGTTGGTTAATACAACAGGAAAAGGTTATGAGAATCATGCGTTGTTAAAAAATGATACTCAGGTTTCTCTCATGTCTTTCAAAGAGGAAAATGATGGATCTTTAGAAGATTTGAGTAAGTTGTCTGTTTCGGATTTACGCATTAAAGTTTCTACTCCAGATATTGTAGAAGAAACTTATGGCCATATGGGGGATTGGTCTGAAGCTACAATTCAAGATGGGGCTCTTGTCATTAATTGGCATCCTACTGGATATAAATTAGATCCGCAAAAAGCTGGTTCTTTGGTATTCAATGCATTATGGGAGGAAGAGGCTGTATTGTCTACTCTAAAAAATGCTCGGATTGCCCATAACCTTACCATTCAGAGAATGGAATTTGATTATTCTACAAATGCTTGGGGATTAGCTTTTAGTAGCTTTAGAGAGCTATCTTCAGAGAAGCTTGTTTCTGTTGATGGATATAGAGGCTCTTATATAGGGGCTTCTGCAGGCATTGATACTCAGTTGATGGAAGATTTTGTTTTGGGAATCAGCACGGCTTCCTTCTTCGGGAAAATGCATAGTCAGAATTTTGATGCAGAGATTTCTCGACATGGTTTTGTTGGTTCGGTCTATACAGGCTTCCTAGCTGGGGCCTGGTTCTTCAAGGGGCAGTACAGTCTTGGCGAAACACATAACGATATGACAACTCGTTACGGGGTTTTGGGAGAATCTAATGCTACTTGGAAGTCTCGAGGAGTACTAGCAGATGCTTTAGTTGAATATCGTAGTTTAGTCGGTCCAGCACGACCTAAATTTTATGCTTTGCATTTTAATCCTTATGTCGAGGTATCTTATGCATCTGCGAAGTTCCCTAGTTTTGTAGAACAAGGAGGAGAAGCTCGTGCTTTTGAAGAAACCTCTTTAACAAACATTACCGTTCCCTTTGGTATGAAATTTGAACTATCTTTTACAAAAGGACAGTTTTCAGAGACTAATTCTCTTGGAATAGGTTGTGCATGGGAAATGTATCGGAAAGTCGAAGGAAGATCTGTAGAGCTACTAGAAGCTGGTTTTGATTGGGAAGGATCTCCTATAGATCTCCCTAAACAAGAGCTGAGAGTGGCTTTAGAAAACAATACGGAATGGAGTTCGTATTTTAGTACAGCTCTAGGAGTAACAGCATTTTGTGGAGGATTTTCTTCTATGGATAATAAACTAGGATACGAAGCGAATGCTGGAATGCGTTTGATTTTCTAG PmpDMSSEKDKKNSCSKFSLSVVAAILASMSGLSNCSDLYAVGSSADHPAYLIPQAGLLLDHIKDIFIGPKDSQD27 PmpD_CHLMUKGQYKLIIGEAGSFQDSNAETLPQKVEHSTLFSVTTPIIVQGIDQQDQVSSQGLVCNFSGDHSEEIFERESFLGIAFLGNGSKDGITLTDIKSSLSGAALYSSDDLIFERIKGDIELSSCSSLERGGACSAQSILTHDCQGLTVKHCAAGVNVEGVSASDHLGEGGGAFSTTSSLSGEKSLYMPAGDIVVATCDGPVCFEGNSAQLANGGAIAASGKVLFVANEKKISFTDNQALSGGAISASSSISFQNCAELVEKSNLAKGVKDKCSLGGGALASLESVVLKDNLGITYEKNQSYSEGGAIFGKDCEIFENRGPVVERDNTAALGGGAILAQQTVAICGNKSGISFEGSKSSFGGAIACGNESSENNSSALGSIDISNNLGDISFLRTLCTTSDLGQTDYQGGGALFAENISLSENAGAITFKDNIVKTFASNGKMLGGGAILASGNVLISKNSGEISFVGNARAPQAIPTRSSDELSFGAQLTQTTSGCSGGGALFGKEVAIVQNATVVFEQNRLQCGEQETHGGGGAVYGMESASIIGNSFVREGNNYAVGNQISGGALLSKKVRLAENTRVDFSRNIATFCGGAVQVSDGSCELINNGYVLFRDNRGQTFGGAISCLKGDVIISGNKDRVEFRDNIVTRPYFEENEEKVETADINSDKQEAEERSLLENIEQSFITATNQTFFLEEEKLPSEAFISAEELSKRRECAGGAIFAKRVYITDNKEPILFSHNFSDVYGGAIFTGSLQETDKQDVVTPEVVISGNDGDVIFSGNAAKHDKHLPDTGGGAICTQNLTISQNNGNVLFLNNFACSGGAVRIEDHGEVLLEAFGGDIIFNGNSSFRAQGSDAIYFAGKDSRIKALNATEGHAIVFQDALVFENIEERKSSGLLVINSQENEGYTGSVRFLGSESKVPQWIHVQQGGLELLHGAILCSYGVKQDPRAKIVLSAGSKLKILDSEQENNAEIGDLEDSVNSEKTPSLWIGKNAQAKVPLVDIHTISIDLASFSSKAQETPEEAPQVIVPKGSCVHSGELSLELVNTTGKGYENHALLKNDTQVSLMSFKEENDGSLEDLSKLSVSDLRIKVSTPDIVEETYGHMGDWSEATIQDGALVINWHPTGYKLDPQKAGSLVFNALWEEEAVLSTLKNARIAHNLTIQRMEFDYSTNAWGLAFSSFRELSSEKLVSVDGYRGSYIGASAGIDTQLMEDFVLGISTASFFGKMHSQNFDAEISRHGFVGSVYTGFLAGAWFFKGQYSLGETHNDMTTRYGVLGESNATWKSRGVLADALVEYRSLVGPARPKFYALHFNPYVEVSYASAKFPSFVEQGGEARAFEETSLTNITVPFGMKFELSFTKGQFSETNSLGIGCAWEMYRKVEGRSVELLEAGFDWEGSPIDLPKQELRVALENNTEWSSYESTALGVTAFCGGFSSMDNKLGYEANAGMRLIF PmpDCATATGGCTATTCTGGCTTCTATGAGTGGTTTATCGAATTGTTCCGATCTGTATGCCGTAGGAAGTTCTGC28 PmpD_EcOpt_dSigAGACCATCCTGCCTACTTGATTCCTCAAGCGGGGTTATTATTGGATCATATTAAGGATATTTTCATTGGCCE. coli codonCTAAAGATAGTCAGGATAAGGGGCAGTATAAGTTGATTATTGGTGAGGCTGGCTCTTTCCAAGATAGTAAToptimized,GCAGAGACTCTGCCTCAAAAGGTAGAGCACAGCACTTTGTTTTCAGTTACAACACCTATTATTGTGCAAGGN-terminalAATTGATCAACAAGATCAGGTCTCTTCGCAGGGATTGGTCTGTAATTTTTCAGGAGATCATTCAGAGGAGAnuclearTTTTTGAGCGCGAATCCTTTTTAGGGATCGCTTTCCTGGGGAATGGTAGCAAGGATGGAATCACGTTAACAlocalizationGATATTAAATCTTCGTTATCTGGTGCTGCCTTGTATTCTTCAGATGATCTGATTTTTGAACGCATTAAGGGsignal removed,AGATATTGAGCTGTCTTCTTGTTCATCTTTAGAACGCGGAGGAGCTTGTTCAGCTCAAAGTATTTTAATTCATGATTGTCAAGGATTAACGGTAAAACATTGTGCCGCAGGGGTGAATGTTGAAGGAGTTAGTGCTAGCGACCATCTGGGATTTGGGGGCGGGGCCTTCTCTACTACAAGTTCTCTGTCTGGAGAGAAGAGTTTGTATATGCCTGCAGGCGATATTGTGGTGGCTACCTGCGATGGTCCTGTGTGTTTCGAAGGAAATAGTGCTCAGTTAGCAAATGGTGGCGCTATTGCCGCTTCTGGTAAAGTTCTGTTTGTAGCTAACGAAAAAAAGATTTCCTTTACAGACAACCAAGCTTTGTCTGGAGGAGCTATTTCTGCATCTTCTAGTATTTCTTTCCAAAATTGTGCTGAGCTGGTGTTCAAGAGTAATCTGGCAAAAGGAGTTAAAGATAAATGTTCTTTGGGAGGAGGTGCTTTAGCCTCTTTAGAATCCGTAGTTTTGAAAGATAATCTGGGTATTACTTATGAAAAAAATCAGTCCTATTCGGAAGGAGGGGCTATTTTTGGGAAGGATTGTGAGATTTTTGAAAACCGCGGGCCTGTTGTATTCCGCGATAATACAGCTGCTTTAGGAGGCGGAGCTATTTTGGCGCAACAAACTGTGGCGATTTGTGGTAATAAGTCTGGAATTTCTTTTGAAGGAAGTAAGTCTAGTTTTGGAGGGGCCATTGCTTGTGGAAATTTCTCTTCTGAGAATAATTCTTCAGCTTTGGGATCAATTGATATCTCTAACAATCTGGGAGATATCTCTTTTCTGCGGACTCTGTGTACTACTTCGGATTTAGGGCAAACGGATTACCAAGGGGGAGGGGCCTTATTCGCTGAAAATATTTCTCTGTCTGAGAATGCTGGTGCAATTACTTTCAAAGACAATATTGTGAAGACATTTGCCTCAAATGGAAAAATGTTGGGTGGAGGGGCAATTTTAGCTTCAGGAAATGTTTTGATTAGCAAAAACTCTGGAGAGATTTCTTTTGTAGGGAATGCCCGTGCTCCTCAGGCTATTCCGACTCGTTCATCTGACGAATTGTCTTTTGGCGCACAATTAACTCAAACTACTTCAGGATGTTCTGGAGGAGGTGCTCTGTTTGGTAAAGAGGTTGCCATTGTTCAAAATGCCACTGTTGTATTCGAGCAAAATCGCTTACAGTGTGGCGAGCAGGAAACACATGGTGGAGGCGGTGCTGTTTATGGTATGGAGAGTGCCTCTATTATTGGAAACTCTTTTGTGCGCTTCGGAAATAATTACGCTGTAGGGAATCAGATTTCTGGAGGTGCTCTGTTATCCAAGAAGGTCCGTTTAGCTGAAAATACACGCGTAGATTTTTCTCGCAATATCGCTACTTTCTGCGGCGGGGCTGTTCAAGTTTCTGATGGAAGTTGCGAATTGATCAACAATGGGTATGTGCTGTTCCGCGATAACCGCGGGCAGACATTTGGTGGGGCTATTTCTTGCTTGAAAGGAGATGTGATCATTTCCGGAAATAAAGATCGCGTTGAGTTTCGCGATAACATTGTGACGCGGCCTTATTTTGAAGAAAATGAAGAAAAAGTTGAGACAGCAGATATTAATTCAGATAAGCAAGAAGCAGAAGAGCGCTCTTTATTAGAGAACATTGAGCAGAGCTTTATTACTGCAACTAATCAGACCTTTTTCTTAGAGGAAGAGAAACTGCCATCAGAAGCTTTTATCTCTGCTGAAGAACTGTCAAAGCGCCGCGAATGTGCTGGTGGGGCGATTTTTGCAAAACGGGTCTACATTACGGATAATAAAGAACCTATCTTGTTTTCGCATAATTTTTCTGATGTTTATGGGGGAGCTATTTTTACGGGTTCTCTGCAGGAAACTGATAAACAAGATGTTGTAACTCCTGAAGTTGTGATTTCAGGCAACGATGGGGATGTCATTTTTTCTGGAAATGCAGCTAAACATGATAAGCATTTACCTGATACAGGTGGTGGAGCCATTTGTACACAGAATTTGACGATTTCCCAAAACAATGGGAATGTCTTGTTCTTGAACAATTTTGCTTGTTCTGGTGGAGCAGTTCGCATTGAGGATCATGGAGAAGTTCTGTTAGAGGCTTTTGGGGGAGATATTATTTTCAATGGAAACTCTTCTTTCCGCGCTCAAGGATCGGATGCGATCTATTTTGCTGGTAAGGACTCTCGCATTAAAGCTTTAAATGCTACTGAAGGACATGCGATTGTGTTCCAAGATGCATTGGTGTTTGAAAATATTGAAGAACGCAAGTCTTCGGGACTGTTGGTGATTAACTCTCAGGAAAATGAGCTCTATACGGGATCTGTCCGCTTTTTAGGATCTGAAAGTAAGGTTCCTCAATGGATTCATGTGCAACAGGGAGGTCTGGAGTTGCTGCATGGAGCTATTTTATGTAGTTATGGGGTTAAACAAGATCCTCGCGCTAAAATTGTATTATCTGCTGGATCTAAATTGAAGATTCTGGATTCAGAGCAAGAAAATAACGCAGAAATTGGAGATCTGGAAGATTCTGTTAATTCAGAAAAAACACCATCTCTGTGGATTGGGAAGAACGCTCAAGCAAAAGTCCCTCTGGTTGATATCCATACTATTTCTATTGATTTAGCATCATTTTCTTCTAAAGCTCAGGAAACCCCTGAGGAAGCTCCACAAGTCATCGTCCCTAAGGGAAGTTGTGTCCACTCGGGAGAGTTAAGTTTGGAGTTGGTTAATACAACAGGAAAAGGTTATGAGAATCATGCGTTGTTAAAAAATGATACTCAGGTTTCTCTGATGTCTTTCAAAGAGGAAAATGATGGATCTTTAGAAGATTTGAGTAAGTTGTCTGTTTCGGATTTACGCATTAAAGTTTCTACTCCAGATATTGTAGAAGAAACTTATGGCCACATGGGGGATTGGTCTGAAGCTACAATTCAAGATGGGGCTCTGGTCATTAATTGGCATCCTACTGGATATAAATTAGATCCGCAAAAAGCTGGTTCTTTGGTATTCAATGCATTATGGGAGGAAGAGGCTGTATTGTCCATGGTGAAAAATGCTCGGATTGCCCATAACCTGACCATTCAGCGCATGGAATTTGATTATTCTACAAATGCTTGGGGATTAGCTTTTAGTAGCTTTCGCGAGCTGTCTTCAGAGAAACTGGTTTCTGTTGATGGATATCGCGGCTCTTATATTGGGGCTTCTGCAGGCATTGATACTCAGTTGATGGAAGATTTTGTTTTGGGAATCAGCACGGCTTCCTTCTTCGGGAAAATGCATAGTCAGAATTTTGATGCAGAGATTTCTCGCCACGGTTTTGTTGGTTCGGTCTATACAGGCTTCCTGGCTGGGGCCTGGTTCTTCAAGGGGCAGTACAGTCTGGGCGAAACACATAACGATATGACAACTCGTTACGGGGTTTTGGGAGAATCTAATGCTACTTGGAAGTCTCGCGGAGTACTGGCAGATGCTTTAGTTGAATATCGTAGTTTAGTCGGTCCAGCACGCCCTAAATTTTATGCTTTGCATTTTAATCCTTATGTCGAGGTATCTTATGCATCTGCGAAGTTCCCTAGTTTTGTAGAACAAGGAGGAGAAGCTCGTGCTTTTGAAGAAACCTCTTTAACAAACATTACCGTTCCGTTTGGTATGAAATTTGAACTGTCTTTTACAAAAGGACAGTTTTCAGAGACTAATTCTCTGGGAATTGGTTGTGCATGGGAAATGTATCGGAAAGTCGAAGGACGCTCTGTAGAGCTGCTGGAAGCTGGTTTTGATTGGGAAGGATCTCCTATTGATCTGCCTAAACAAGAGCTGCGCGTGGCTTTAGAAAACAATACGGAATGGAGTTCGTATTTTAGTACAGCTCTGGGAGTAACAGCATTTTGTGGAGGATTTTCTTCTATGGATAATAAACTGGGATACGAAGCGAATGCTGGAATGCGTTTGATTTTCtaaGGATCC PmpDMAILASMSGLSNCSDLYAVGSSADHPAYLIPQAGLLLDHIKDIFIGPKDSQDKGQYKLIIGEAGSFQDSNA29 PmpD_ExOpt_dSigETLPQKVEHSTLFSVTTPIIVQGIDQQDQVSSQGLVCNFSGDHSEEIFERESFLGIAFLGNGSKDGITLTDE. coli codonIKSSLSGAALYSSDDLIFERIKGDIELSSCSSLERGGACSAQSILIHDCQGLTVKHCAAGVNVEGVSASDHoptimized,LGEGGGAFSTTSSLSGEKSLYMPAGDIVVATCDGPVCFEGNSAQLANGGAIAASGKVLFVANEKKISFTDNN-terminalQALSGGAISASSSISFQNCAELVEKSNLAKGVKDKCSLGGGALASLESVVLKDNLGITYEKNQSYSEGGAInuclearFGKDCEIFENRGPVVFRDNTAALGGGAILAQQTVAICGNKSGISFEGSKSSEGGAIACGNESSENNSSALGlocalizationSIDISNNLGDISFLRTLCTTSDLGQTDYQGGGALFAENISLSENAGAITFKDNIVKTFASNGKMLGGGAILsignal removedASGNVLISKNSGEISFVGNARAPQAIPTRSSDELSFGAQLTQTTSGCSGGGALFGKEVAIVQNATVVFEQNRLQCGEQETHGGGGAVYGMESASIIGNSFVREGNNYAVGNQISGGALLSKKVRLAENTRVDFSRNIATFCGGAVQVSDGSCELINNGYVLFRDNRGQTFGGAISCLKGDVIISGNKDRVEFRDNIVTRPYFEENEEKVETADINSDKQEAEERSLLENIEQSFITATNQTFFLEEEKLPSEAFISAEELSKRRECAGGAIFAKRVYITDNKEPILFSHNFSDVYGGAIFTGSLQETDKQDVVTPEVVISGNDGDVIFSGNAAKHDKHLPDTGGGAICTQNLTISQNNGNVLFLNNFACSGGAVRIEDHGEVLLEAFGGDIIFNGNSSFRAQGSDAIYFAGKDSRIKALNATEGHAIVFQDALVFENIEERKSSGLLVINSQENELYTGSVRFLGSESKVPQWIHVQQGGLELLHGAILCSYGVKQDPRAKIVLSAGSKLKILDSEQENNAEIGDLEDSVNSEKTPSLWIGKNAQAKVPLVDIHTISIDLASFSSKAQETPEEAPQVIVPKGSCVHSGELSLELVNTTGKGYENHALLKNDTQVSLMSFKEENDGSLEDLSKLSVSDLRIKVSTPDIVEETYGHMGDWSEATIQDGALVINWHPTGYKLDPQKAGSLVFNALWEEEAVLSMVKNARIAHNLTIQRMEFDYSTNAWGLAFSSFRELSSEKLVSVDGYRGSYIGASAGIDTQLMEDFVLGISTASFFGKMHSQNFDAEISRHGFVGSVYTGFLAGAWFFKGQYSLGETHNDMTTRYGVLGESNATWKSRGVLADALVEYRSLVGPARPKFYALHFNPYVEVSYASAKFPSFVEQGGEARAFEETSLTNITVPFGMKFELSFTKGQFSETNSLGIGCAWEMYRKVEGRSVELLEAGFDWEGSPIDLPKQELRVALENNTEWSSYESTALGVTAFCGGFSSMDNKLGYEANAGMRLIF PmpDCATATGGCTATTCTGGCTTCTATGAGTGGTTTATCGAATTGTTCCGATCTGTATGCCGTAGGAAGTTCTGC30 PmpD_EcOpt_dSig_AGACCATCCTGCCTACTTGATTCCTCAAGCGGGGTTATTATTGGATCATATTAAGGATATTTTCATTGGCCdPMPCTAAAGATAGTCAGGATAAGGGGCAGTATAAGTTGATTATTGGTGAGGCTGGCTCTTTCCAAGATAGTAATE. coli codonGCAGAGACTCTGCCTCAAAAGGTAGAGCACAGCACTTTGTTTTCAGTTACAACACCTATTATTGTGCAAGGoptimized,AATTGATCAACAAGATCAGGTCTCTTCGCAGGGATTGGTCTGTAATTTTTCAGGAGATCATTCAGAGGAGAN-terminalTTTTTGAGCGCGAATCCTTTTTAGGGATCGCTTTCCTGGGGAATGGTAGCAAGGATGGAATCACGTTAACAnuclearGATATTAAATCTTCGTTATCTGGTGCTGCCTTGTATTCTTCAGATGATCTGATTTTTGAACGCATTAAGGGlocalizationAGATATTGAGCTGTCTTCTTGTTCATCTTTAGAACGCGGAGGAGCTTGTTCAGCTCAAAGTATTTTAATTCsignal removed,ATGATTGTCAAGGATTAACGGTAAAACATTGTGCCGCAGGGGTGAATGTTGAAGGAGTTAGTGCTAGCGACadhesion domainCATCTGGGATTTGGGGGCGGGGCCTTCTCTACTACAAGTTCTCTGTCTGGAGAGAAGAGTTTGTATATGCC(ChlamPMP_M)TGCAGGCGATATTGTGGTGGCTACCTGCGATGGTCCTGTGTGTTTCGAAGGAAATAGTGCTCAGTTAGCAAremovedATGGTGGCGCTATTGCCGCTTCTGGTAAAGTTCTGTTTGTAGCTAACGAAAAAAAGATTTCCTTTACAGACAACCAAGCTTTGTCTGGAGGAGCTATTTCTGCATCTTCTAGTATTTCTTTCCAAAATTGTGCTGAGCTGGTGTTCAAGAGTAATCTGGCAAAAGGAGTTAAAGATAAATGTTCTTTGGGAGGAGGTGCTTTAGCCTCTTTAGAATCCGTAGTTTTGAAAGATAATCTGGGTATTACTTATGAAAAAAATCAGTCCTATTCGGAAGGAGGGGCTATTTTTGGGAAGGATTGTGAGATTTTTGAAAACCGCGGGCCTGTTGTATTCCGCGATAATACAGCTGCTTTAGGAGGCGGAGCTATTTTGGCGCAACAAACTGTGGCGATTTGTGGTAATAAGTCTGGAATTTCTTTTGAAGGAAGTAAGTCTAGTTTTGGAGGGGCCATTGCTTGTGGAAATTTCTCTTCTGAGAATAATTCTTCAGCTTTGGGATCAATTGATATCTCTAACAATCTGGGAGATATCTCTTTTCTGCGGACTCTGTGTACTACTTCGGATTTAGGGCAAACGGATTACCAAGGGGGAGGGGCCTTATTCGCTGAAAATATTTCTCTGTCTGAGAATGCTGGTGCAATTACTTTCAAAGACAATATTGTGAAGACATTTGCCTCAAATGGAAAAATGTTGGGTGGAGGGGCAATTTTAGCTTCAGGAAATGTTTTGATTAGCAAAAACTCTGGAGAGATTTCTTTTGTAGGGAATGCCCGTGCTCCTCAGGCTATTCCGACTCGTTCATCTGACGAATTGTCTTTTGGCGCACAATTAACTCAAACTACTTCAGGATGTTCTGGAGGAGGTGCTCTGTTTGGTAAAGAGGTTGCCATTGTTCAAAATGCCACTGTTGTATTCGAGCAAAATCGCTTACAGTGTGGCGAGCAGGAAACACATGGTGGAGGCGGTGCTGTTTATGGTATGGAGAGTGCCTCTATTATTGGAAACTCTTTTGTGCGCTTCGGAAATAATTACGCTGTAGGGAATCAGATTTCTGGAGGTGCTCTGTTATCCAAGAAGGTCCGTTTAGCTGAAAATACACGCGTAGATTTTTCTCGCAATATCGCTACTTTCTGCGGCGGGGCTGTTCAAGTTTCTGATGGAAGTTGCGAATTGATCAACAATGGGTATGTGCTGTTCCGCGATAACCGCGGGCAGACATTTGGTGGGGCTATTTCTTGCTTGAAAGGAGATGTGATCATTTCCGGAAATAAAGATCGCGTTGAGTTTCGCGATAACATTGTGACGCGGCCTTATTTTGAAGAAAATGAAGAAAAAGTTGAGACAGCAGATATTAATTCAGATAAGCAAGAAGCAGAAGAGCGCTCTTTATTAGAGAACATTGAGCAGAGCTTTATTACTGCAACTAATCAGACCTTTTTCTTAGAGGAAGAGAAACTGCCATCAGAAGCTTTTATCTCTGCTGAAGAACTGTCAAAGCGCCGCGAATGTGCTGGTGGGGCGATTTTTGCAAAACGGGTCTACATTACGGATAATAAAGAACCTATCTTGTTTTCGCATAATTTTTCTGATGTTTATGGGGGAGCTATTTTTACGGGTTCTCTGCAGGAAACTGATAAACAAGATGTTGTAACTCCTGAAGTTGTGATTTCAGGCAACGATGGGGATGTCATTTTTTCTGGAAATGCAGCTAAACATGATAAGCATTTACCTGATACAGGTGGTGGAGCCATTTGTACACAGAATTTGACGATTTCCCAAAACAATGGGAATGTCTTGTTCTTGAACAATTTTGCTTGTTCTGGTGGAGCAGTTCGCATTGAGGATCATGGAGAAGTTCTGTTAGAGGCTTTTGGGGGAGATATTATTTTCAATGGAAACTCTTCTTTCCGCGCTCAAGGATCGGATGCGATCTATTTTGCTGGTAAGGACTCTCGCATTAAAGCTTTAAATGCTACTGAAGGACATGCGATTGTGTTCCAAGATGCATTGGTGTTTGAAAATATTGAAGAACGCAAGTCTTCGGGACTGTTGGTGATTAACTCTCAGGAAAATGAGCTCTATACGGGATCTGTCCGCTTTTTAGGATCTGAAAGTAAGGTTCCTCAATGGATTCATGTGCAACAGACTGGATATAAATTAGATCCGCAAAAAGCTGGTTCTTTGGTATTCAATGCATTATGGGAGGAAGAGGCTGTATTGTCCATGGTGAAAAATGCTCGGATTGCCCATAACCTGACCATTCAGCGCATGGAATTTGATTATTCTACAAATGCTTGGGGATTAGCTTTTAGTAGCTTTCGCGAGCTGTCTTCAGAGAAACTGGTTTCTGTTGATGGATATCGCGGCTCTTATATTGGGGCTTCTGCAGGCATTGATACTCAGTTGATGGAAGATTTTGTTTTGGGAATCAGCACGGCTTCCTTCTTCGGGAAAATGCATAGTCAGAATTTTGATGCAGAGATTTCTCGCCACGGTTTTGTTGGTTCGGTCTATACAGGCTTCCTGGCTGGGGCCTGGTTCTTCAAGGGGCAGTACAGTCTGGGCGAAACACATAACGATATGACAACTCGTTACGGGGTTTTGGGAGAATCTAATGCTACTTGGAAGTCTCGCGGAGTACTGGCAGATGCTTTAGTTGAATATCGTAGTTTAGTCGGTCCAGCACGCCCTAAATTTTATGCTTTGCATTTTAATCCTTATGTCGAGGTATCTTATGCATCTGCGAAGTTCCCTAGTTTTGTAGAACAAGGAGGAGAAGCTCGTGCTTTTGAAGAAACCTCTTTAACAAACATTACCGTTCCGTTTGGTATGAAATTTGAACTGTCTTTTACAAAAGGACAGTTTTCAGAGACTAATTCTCTGGGAATTGGTTGTGCATGGGAAATGTATCGGAAAGTCGAAGGACGCTCTGTAGAGCTGCTGGAAGCTGGTTTTGATTGGGAAGGATCTCCTATTGATCTGCCTAAACAAGAGCTGCGCGTGGCTTTAGAAAACAATACGGAATGGAGTTCGTATTTTAGTACAGCTCTGGGAGTAACAGCATTTTGTGGAGGATTTTCTTCTATGGATAATAAACTGGGATACGAAGCGAATGCTGGAATGCGTTTGATTTTCtaaGGATCC PmpDMAILASMSGLSNCSDLYAVGSSADHPAYLIPQAGLLLDHIKDIFIGPKDSQDKGQYKLIIGEAGSFQDSNA31 PmpD_EcOpt_dSig_ETLPQKVEHSTLFSVTTPIIVQGIDQQDQVSSQGLVCNFSGDHSEEIFERESFLGIAFLGNGSKDGITLTDdPMPIKSSLSGAALYSSDDLIFERIKGDIELSSCSSLERGGACSAQSILIHDCQGLTVKHCAAGVNVEGVSASDHE. coli codonLGFGGGAFSTTSSLSGEKSLYMPAGDIVVATCDGPVCFEGNSAQLANGGAIAASGKVLFVANEKKISFTDNoptimized,QALSGGAISASSSISFQNCAELVEKSNLAKGVKDKCSLGGGALASLESVVLKDNLGITYEKNQSYSEGGAIN-terminalFGKDCEIFENRGPVVERDNTAALGGGAILAQQTVAICGNKSGISFEGSKSSFGGAIACGNFSSENNSSALGnuclearSIDISNNLGDISFLRTLCTTSDLGQTDYQGGGALFAENISLSENAGAITFKDNIVKTFASNGKMLGGGAILlocalizationASGNVLISKNSGEISFVGNARAPQAIPTRSSDELSFGAQLTQTTSGCSGGGALFGKEVAIVQNATVVFEQNsignal removed,RLQCGEQETHGGGGAVYGMESASIIGNSFVREGNNYAVGNQISGGALLSKKVRLAENTRVDFSRNIATFCGadhesion domainGAVQVSDGSCELINNGYVLFRDNRGQTFGGAISCLKGDVIISGNKDRVEFRDNIVTRPYFEENEEKVETAD(ChlamPMP_M)INSDKQEAEERSLLENIEQSFITATNQTFFLEEEKLPSEAFISAEELSKRRECAGGAIFAKRVYITDNKEPremovedILFSHNFSDVYGGAIFTGSLQETDKQDVVTPEVVISGNDGDVIFSGNAAKHDKHLPDTGGGAICTQNLTISQNNGNVLFLNNFACSGGAVRIEDHGEVLLEAFGGDIIFNGNSSFRAQGSDAIYFAGKDSRIKALNATEGHAIVFQDALVFENIEERKSSGLLVINSQENELYTGSVRFLGSESKVPQWIHVQQTGYKLDPQKAGSLVFNALWEEEAVLSMVKNARIAHNLTIQRMEFDYSTNAWGLAFSSFRELSSEKLVSVDGYRGSYIGASAGIDTQLMEDFVLGISTASFFGKMHSQNFDAEISRHGFVGSVYTGFLAGAWFFKGQYSLGETHNDMTTRYGVLGESNATWKSRGVLADALVEYRSLVGPARPKEYALHENPYVEVSYASAKEPSFVEQGGEARAFEETSLTNITVPFGMKFELSFTKGQFSETNSLGIGCAWEMYRKVEGRSVELLEAGFDWEGSPIDLPKQELRVALENNTEWSSYESTALGVTAFCGGFSSMDNKLGYEANAGMRLIF PmpEATGAAAAAACTGTTCTTTTTTGTCCTTATTGGAAGCTCTATACTGGGATTTACTCGAGAAGTCCCTCCTTC32 TC_RS01325-GATTCTTTTAAAGCCTATACTAAATCCATACCATATGACCGGGTTATTTTTTCCCAAGGTTAATTTGCTTG124631 (reversed)GAGACACACATAATCTCACTGATTACCATTTGGATAATCTAAAATGCATTCTGGCTTGCCTACAAAGAACTOriginal databaseCCTTATGAAGGAGCTGCTTTCACAGTAACCGATTACTTAGGTTTTTCAGATACACAAAAGGATGGTATTTTsequenceTTGTTTTAAAAATCTTACTCCAGAGAGTGGAGGGGTTATTGGTTCCCCAACTCAAAACACTCCTACTATAAAAATTCATAATACAATCGGCCCCGTTCTTTTCGAAAATAATACCTGTCATAGACTGTGGACACAGACCGATCCCGAAAATGAAGGAAACAAAGCACGCGAAGGCGGGGCAATTCATGCTGGGGACGTTTACATAAGCAATAACCAGAACCTTGTCGGATTCATAAAGAACTTTGCTTATGTTCAAGGTGGAGCTATTAGTGCTAATACTTTTGCCTATAAAGAAAATAAATCGAGCTTTCTTTGCCTAAATAACTCTTGTATACAAACTAAGACGGGAGGGAAAGGTGGTGCTATTTACGTTAGTACGAGCTGCTCTTTCGAGAACAATAACAAGGATCTGCTTTTCATCCAAAACTCCGGCTGTGCAGGAGGAGCTATCTTCTCTCCAACCTGTTCTCTAATAGGAAACCAAGGAGATATTGTTTTTTACAGCAACCACGGTTTTAAAAATGTTGATAATGCAACTAACGAATCTGGGGATGGAGGAGCTATTAAAGTAACTACCCGCTTGGACATCACCAATAATGGTAGTCAAATCTTTTTTTCTGATAATATCTCAAGAAATTTTGGAGGAGCTATTCATGCTCCTTGTCTTCATCTTGTTGGTAATGGGCCAACCTATTTTACAAACAATATAGCTAATCACACAGGTGGGGCTATTTATATAACAGGAACAGAAACCTCAAAGATTTCTGCAGATCACCATGCTATTATTTTTGATAATAACATTTCTGCAAACGCCACCAATGCGGACGGATCTAGCAGCAACACTAATCCTCCTCACAGAAATGCGATCACTATGGACAATTCCGCTGGAGGAATAGAACTTGGTGCAGGGAAGAGCCAGAATCTTATTTTCTATGATCCTATTCAAGTGACGAATGCTGGAGTTACCGTAGACTTCAATAAGGATGCCTCCCAAACCGGATGTGTAGTTTTCTCTGGAGCGACTGTCCTTTCTGCAGATATTTCTCAGGCTAATTTGCAAACTAAAACACCTGCAACGCTTACTCTCAGTCACGGTCTTCTGTGTATCGAAGATCGTGCTCAGCTCACAGTGAACAATTTTACACAAACAGGAGGGATTGTAGCCTTAGGAAATGGAGCAGTTTTAAGCAGCTACCAACACAGCACTACAGACGCCACTCAAACTCCCCCTACAACCACCACTACAGATGCTTCCGTAACTCTTAATCACATTGGATTAAATCTCCCCTCTATTCTTAAGGATGGAGCAGAGATGCCTCTATTATGGGTAGAACCTATAAGCACAACTCAAGGTAACACTACAACATATACGTCAGATACCGCGGCTTCCTTCTCATTAAATGGAGCCACACTCTCTCTCATTGATGAAGATGGAAATTCTCCCTATGAAAACACGGACCTCTCTCGTGCATTGTACGCTCAACCTATGCTAGCAATTTCTGAGGCCAGTGATAACCAATTGCAATCCGAAAGCATGGACTTTTCTAAAGTTAATGTTCCTCACTATGGATGGCAAGGACTTTGGACCTGGGGGTGGGCAAAAACTGAAAATCCAACAACAACTCCTCCAGCAACAATTACTGATCCGAAAAAAGCTAATCAGTTTCATAGAACTTTATTATTAACGTGGCTCCCTGCTGGTTATATCCCCAGCCCTAAACATAAAAGCCCTTTAATAGCTAATACCTTGTGGGGGAATATACTTTTTGCAACGGAAAACTTAAAAAATAGCTCAGGGCAAGAACTTCTTGATCGTCCTTTCTGGGGAATTACAGGAGGGGGCTTGGGGATGATGGTCTATCAAGAACCTAGAAAAGACCATCCTGGATTCCACATGCATACCTCCGGATATTCAGCAGGAATGATTACAGGAAACACACATACCTTCTCATTACGATTCAGCCAGTCCTATACAAAACTCAATGAACGTTATGCCAAGAACTATGTGTCTTCTAAAAATTACTCTTGCCAAGGGGAAATGCTTTTGTCCTTACAAGAAGGACTCATGCTGACTAAACTAATTGGTCTCTATAGTTATGGGAATCACAACAGCCACCATTTCTATACCCAAGGAGAAGACCTATCGTCTCAAGGGGAGTTCCATAGTCAGACTTTTGGAGGGGCTGTCTTTTTTGATCTACCTCTGAAACCTTTTGGAAGAACACACATACTTACAGCTCCTTTCTTAGGTGCCATTGGTATGTATTCTAAGCTGTCTAGCTTTACAGAAGTAGGAGCCTATCCAAGAACCTTTATTACAGAAACGCCTTTAATCAATGTCCTGATTCCTATCGGAGTAAAAGGTAGCTTCATGAATGCCACCCATAGACCTCAGGCCTGGACTGTAGAGCTTGCTTACCAACCTGTTCTTTACAGACAAGAACCTAGTATCTCTACCCAATTACTCGCTGGTAAAGGTATGTGGTTTGGGCATGGAAGTCCTGCATCTCGCCACGCTCTAGCTTATAAAATTTCACAGAAAACACAGCTTTTGCGATTTGCAACACTTCAACTCCAGTATCACGGATACTATTCGTCTTCCACTTTCTGTAATTATCTGAATGGAGAGGTATCTTTACGTTTCTAA PmpEMKKLFFFVLIGSSILGFTREVPPSILLKPILNPYHMTGLFFPKVNLLGDTHNLTDYHLDNLKCILACLQRT33 PMPE_CHLMUPYEGAAFTVTDYLGFSDTQKDGIFCFKNLTPESGGVIGSPTQNTPTIKIHNTIGPVLFENNTCHRLWTQTDPENEGNKAREGGAIHAGDVYISNNQNLVGFIKNFAYVQGGAISANTFAYKENKSSFLCLNNSCIQTKTGGKGGAIYVSTSCSFENNNKDLLFIQNSGCAGGAIFSPTCSLIGNQGDIVEYSNHGEKNVDNATNESGDGGAIKVTTRLDITNNGSQIFFSDNISRNFGGAIHAPCLHLVGNGPTYFTNNIANHTGGAIYITGTETSKISADHHAIIFDNNISANATNADGSSSNTNPPHRNAITMDNSAGGIELGAGKSQNLIFYDPIQVTNAGVTVDFNKDASQTGCVVFSGATVLSADISQANLQTKTPATLTLSHGLLCIEDRAQLTVNNFTQTGGIVALGNGAVLSSYQHSTTDATQTPPTTTTTDASVTLNHIGLNLPSILKDGAEMPLLWVEPISTTQGNTTTYTSDTAASFSLNGATLSLIDEDGNSPYENTDLSRALYAQPMLAISEASDNQLQSESMDFSKVNVPHYGWQGLWTWGWAKTENPTTTPPATITDPKKANQFHRTLLLTWLPAGYIPSPKHKSPLIANTLWGNILFATENLKNSSGQELLDRPFWGITGGGLGMMVYQEPRKDHPGFHMHTSGYSAGMITGNTHTFSLRFSQSYTKLNERYAKNYVSSKNYSCQGEMLLSLQEGLMLTKLIGLYSYGNHNSHHFYTQGEDLSSQGEFHSQTFGGAVFFDLPLKPFGRTHILTAPFLGAIGMYSKLSSFTEVGAYPRTFITETPLINVLIPIGVKGSFMNATHRPQAWTVELAYQPVLYRQEPSISTQLLAGKGMWFGHGSPASRHALAYKISQKTQLLRFATLQLQYHGYYSSSTFCNYLNGEVSLRF PmpECATATGCGCGAAGTCCCTCCTTCGATTCTGTTAAAGCCTATTCTGAATCCATACCACATGACCGGGTTATT34 PmpE_EcOpt_dSigTTTTCCGAAGGTTAATTTGCTGGGAGACACACATAATCTGACTGATTACCATTTGGATAATCTGAAATGCAE. coli codonTTCTGGCTTGCCTGCAACGCACTCCTTATGAAGGAGCTGCTTTCACAGTAACCGATTACTTAGGTTTTTCAoptimized,GATACACAAAAGGATGGTATTTTTTGTTTTAAAAATCTGACTCCAGAGAGTGGAGGGGTTATTGGTTCCCCN-terminalAACTCAAAACACTCCTACTATTAAAATTCATAATACAATCGGCCCGGTTCTGTTCGAAAATAATACCTGTCnuclearATCGCCTGTGGACACAGACCGATCCGGAAAATGAAGGAAACAAAGCACGCGAAGGCGGGGCAATTCATGCTlocalizationGGGGACGTTTACATTAGCAATAACCAGAACCTGGTCGGATTCATTAAGAACTTTGCTTATGTTCAAGGTGGsignal removedAGCTATTAGTGCTAATACTTTTGCCTATAAAGAAAATAAATCGAGCTTTCTGTGCCTGAATAACTCTTGTATTCAAACTAAGACGGGAGGGAAAGGTGGTGCTATTTACGTTAGTACGAGCTGCTCTTTCGAGAACAATAACAAGGATCTGCTGTTCATCCAAAACTCCGGCTGTGCAGGAGGAGCTATCTTCTCTCCAACCTGTTCTCTGATTGGAAACCAAGGAGATATTGTTTTTTACAGCAACCACGGTTTTAAAAATGTTGATAATGCAACTAACGAATCTGGGGATGGAGGAGCTATTAAAGTAACTACCCGCTTGGACATCACCAATAATGGTAGTCAAATCTTTTTTTCTGATAATATCTCACGCAATTTTGGAGGAGCTATTCATGCTCCTTGTCTGCATCTGGTTGGTAATGGGCCAACCTATTTTACAAACAATATTGCTAATCACACAGGTGGGGCTATTTATATTACAGGAACAGAAACCTCAAAGATTTCTGCAGATCACCATGCTATTATTTTTGATAATAACATTTCTGCAAACGCCACCAATGCGGACGGATCTAGCAGCAACACTAATCCTCCTCACCGCAATGCGATCACTATGGACAATTCCGCTGGAGGAATTGAACTGGGTGCAGGGAAGAGCCAGAATCTGATTTTCTATGATCCTATTCAAGTGACGAATGCTGGAGTTACCGTAGACTTCAATAAGGATGCCTCCCAAACCGGATGTGTAGTTTTCTCTGGAGCGACTGTCCTGTCTGCAGATATTTCTCAGGCTAATTTGCAAACTAAAACACCTGCAGAGCTCACTCTGAGTCACGGTCTGCTGTGTATCGAAGATCGTGCTCAGCTGACAGTGAACAATTTTACACAAACAGGAGGGATTGTAGCCTTAGGAAATGGAGCAGTTTTAAGCAGCTACCAACACAGCACTACAGACGCCACTCAAACTCCGCCTACAACCACCACTACAGATGCTTCCGTAACTCTGAATCACATTGGATTAAATCTGCCGTCTATTCTGAAGGATGGAGCAGAGATGCCTCTGTTATGGGTAGAACCTATTAGCACAACTCAAGGTAACACTACAACATATACGTCAGATACCGCGGCTTCCTTCTCATTAAATGGAGCCACACTGTCTCTGATTGATGAAGATGGAAATTCTCCGTATGAAAACACGGACCTGTCTCGTGCATTGTACGCTCAACCTATGCTGGCAATTTCTGAGGCCAGTGATAACCAATTGCAATCCGAAAGCATGGACTTTTCTAAAGTTAATGTTCCTCACTATGGATGGCAAGGACTGTGGACCTGGGGGTGGGCAAAAACTGAAAATCCAACAACAACTCCTCCAGCAACAATTACTGATCCGAAAAAAGCTAATCAGTTTCATCGCACTTTATTATTAACGTGGCTGCCTGCTGGTTATATCCCGAGCCCTAAACATAAAAGCCCTTTAATTGCTAATACCTTGTGGGGGAATATTGCCATGGCAACGGAAAACTTAAAAAATAGCTCAGGGCAAGAACTGCTGGATCGTCCTTTCTGGGGAATTACAGGAGGGGGCTTGGGGATGATGGTCTATCAAGAACCTCGCAAAGACCATCCTGGATTCCACATGCATACCTCCGGATATTCAGCAGGAATGATTACAGGAAACACACATACCTTCTCATTACGCTTCAGCCAGTCCTATACAAAACTGAATGAACGTTATGCCAAGAACTATGTGTCTTCTAAAAATTACTCTTGCCAAGGGGAAATGCTGTTGTCCTTACAAGAAGGACTGATGCTGACTAAACTGATTGGTCTGTATAGTTATGGGAATCACAACAGCCACCATTTCTATACCCAAGGAGAAGACCTGTCGTCTCAAGGGGAGTTCCATAGTCAGACTTTTGGAGGGGCTGTCTTTTTTGATCTGCCTCTGAAACCTTTTGGACGCACACACATTCTGACAGCTCCTTTCTTAGGTGCCATTGGTATGTATTCTAAGCTGTCTAGCTTTACAGAAGTAGGAGCCTATCCACGCACCTTTATTACAGAAACGCCTTTAATCAATGTCCTGATTCCTATCGGAGTAAAAGGTAGCTTCATGAATGCCACCCATCGCCCTCAGGCCTGGACTGTAGAGCTGGCTTACCAACCTGTTCTGTACCGCCAAGAACCTAGTATCTCTACCCAATTACTGGCTGGTAAAGGTATGTGGTTTGGGCATGGAAGTCCTGCATCTCGCCACGCTCTGGCTTATAAAATTTCACAGAAAACACAGCTGTTGCGCTTTGCAACACTGCAACTGCAGTATCACGGATACTATTCGTCTTCCACTTTCTGTAATTATCTGAATGGAGAGGTATCTTTACGTTTCtaaGGATCC PmpEMREVPPSILLKPILNPYHMTGLFFPKVNLLGDTHNLTDYHLDNLKCILACLQRTPYEGAAFTVTDYLGFSD35 PmpE_EcOpt_dSigTQKDGIFCFKNLTPESGGVIGSPTQNTPTIKIHNTIGPVLFENNTCHRLWTQTDPENEGNKAREGGAIHAGE. coli codonDVYISNNQNLVGFIKNFAYVQGGAISANTFAYKENKSSFLCLNNSCIQTKTGGKGGAIYVSTSCSFENNNKoptimized,DLLFIQNSGCAGGAIFSPTCSLIGNQGDIVFYSNHGFKNVDNATNESGDGGAIKVTTRLDITNNGSQIFFSN-terminalDNISRNFGGAIHAPCLHLVGNGPTYFTNNIANHTGGAIYITGTETSKISADHHAIIFDNNISANATNADGSnuclearSSNTNPPHRNAITMDNSAGGIELGAGKSQNLIFYDPIQVTNAGVTVDFNKDASQTGCVVFSGATVLSADISlocalizationQANLQTKTPAELTLSHGLLCIEDRAQLTVNNFTQTGGIVALGNGAVLSSYQHSTTDATQTPPTTTTTDASVsignal removedTLNHIGLNLPSILKDGAEMPLLWVEPISTTQGNTTTYTSDTAASFSLNGATLSLIDEDGNSPYENTDLSRALYAQPMLAISEASDNQLQSESMDFSKVNVPHYGWQGLWTWGWAKTENPTTTPPATITDPKKANQFHRTLLLTWLPAGYIPSPKHKSPLIANTLWGNIAMATENLKNSSGQELLDRPFWGITGGGLGMMVYQEPRKDHPGFHMHTSGYSAGMITGNTHTFSLRFSQSYTKLNERYAKNYVSSKNYSCQGEMLLSLQEGLMLTKLIGLYSYGNHNSHHFYTQGEDLSSQGEFHSQTFGGAVFFDLPLKPFGRTHILTAPFLGAIGMYSKLSSFTEVGAYPRTFITETPLINVLIPIGVKGSFMNATHRPQAWTVELAYQPVLYRQEPSISTQLLAGKGMWFGHGSPASRHALAYKISQKTQLLRFATLQLQYHGYYSSSTFCNYLNGEVSLRF PmpECATATGCGCGAAGTCCCTCCTTCGATTCTGTTAAAGCCTATTCTGAATCCATACCACATGACCGGGTTATT36 E. coli codonTTTTCCGAAGGTTAATTTGCTGGGAGACACACATAATCTGACTGATTACCATTTGGATAATCTGAAATGCAoptimized,TTCTGGCTTGCCTGCAACGCACTCCTTATGAAGGAGCTGCTTTCACAGTAACCGATTACTTAGGTTTTTCAN-terminalGATACACAAAAGGATGGTATTTTTTGTTTTAAAAATCTGACTCCAGAGAGTGGAGGGGTTATTGGTTCCCCnuclearAACTCAAAACACTCCTACTATTAAAATTCATAATACAATCGGCCCGGTTCTGTTCGAAAATAATACCTGTClocalizationATCGCCTGTGGACACAGACCGATCCGGAAAATGAAGGAAACAAAGCACGCGAAGGCGGGGCAATTCATGCTsignal removed,GGGGACGTTTACATTAGCAATAACCAGAACCTGGTCGGATTCATTAAGAACTTTGCTTATGTTCAAGGTGGadhesion domainAGCTATTAGTGCTAATACTTTTGCCTATAAAGAAAATAAATCGAGCTTTCTGTGCCTGAATAACTCTTGTA(ChlamPMP_M)TTCAAACTAAGACGGGAGGGAAAGGTGGTGCTATTTACGTTAGTACGAGCTGCTCTTTCGAGAACAATAACremovedAAGGATCTGCTGTTCATCCAAAACTCCGGCTGTGCAGGAGGAGCTATCTTCTCTCCAACCTGTTCTCTGATTGGAAACCAAGGAGATATTGTTTTTTACAGCAACCACGGTTTTAAAAATGTTGATAATGCAACTAACGAATCTGGGGATGGAGGAGCTATTAAAGTAACTACCCGCTTGGACATCACCAATAATGGTAGTCAAATCTTTTTTTCTGATAATATCTCACGCAATTTTGGAGGAGCTATTCATGCTCCTTGTCTGCATCTGGTTGGTAATGGGCCAACCTATTTTACAAACAATATTGCTAATCACACAGGTGGGGCTATTTATATTACAGGAACAGAAACCTCAAAGATTTCTGCAGATCACCATGCTATTATTTTTGATAATAACATTTCTGCAAACGCCACCAATGCGGACGGATCTAGCAGCAACACTAATCCTCCTCACCGCAATGCGATCACTATGGACAATTCCGCTGGAGGAATTGAACTGGGTGCAGGGAAGAGCCAGAATCTGATTTTCTATGATCCTATTCAAGTGACGAATGCTGGAGTTACCGTAGACTTCAATAAGGATGCCTCCCAAACCGGATGTGTAGTTTTCTCTGGAGCGACTGTCCTGTCTGCAGATATTTCTCAGGCTAATTTGCAAACTAAAACACCTGCAGAGCTCACTCTGAGTCACCCTGCTGGTTATATCCCGAGCCCTAAACATAAAAGCCCTTTAATTGCTAATACCTTGTGGGGGAATATTGCCATGGCAACGGAAAACTTAAAAAATAGCTCAGGGCAAGAACTGCTGGATCGTCCTTTCTGGGGAATTACAGGAGGGGGCTTGGGGATGATGGTCTATCAAGAACCTCGCAAAGACCATCCTGGATTCCACATGCATACCTCCGGATATTCAGCAGGAATGATTACAGGAAACACACATACCTTCTCATTACGCTTCAGCCAGTCCTATACAAAACTGAATGAACGTTATGCCAAGAACTATGTGTCTTCTAAAAATTACTCTTGCCAAGGGGAAATGCTGTTGTCCTTACAAGAAGGACTGATGCTGACTAAACTGATTGGTCTGTATAGTTATGGGAATCACAACAGCCACCATTTCTATACCCAAGGAGAAGACCTGTCGTCTCAAGGGGAGTTCCATAGTCAGACTTTTGGAGGGGCTGTCTTTTTTGATCTGCCTCTGAAACCTTTTGGACGCACACACATTCTGACAGCTCCTTTCTTAGGTGCCATTGGTATGTATTCTAAGCTGTCTAGCTTTACAGAAGTAGGAGCCTATCCACGCACCTTTATTACAGAAACGCCTTTAATCAATGTCCTGATTCCTATCGGAGTAAAAGGTAGCTTCATGAATGCCACCCATCGCCCTCAGGCCTGGACTGTAGAGCTGGCTTACCAACCTGTTCTGTACCGCCAAGAACCTAGTATCTCTACCCAATTACTGGCTGGTAAAGGTATGTGGTTTGGGCATGGAAGTCCTGCATCTCGCCACGCTCTGGCTTATAAAATTTCACAGAAAACACAGCTGTTGCGCTTTGCAACACTGCAACTGCAGTATCACGGATACTATTCGTCTTCCACTTTCTGTAATTATCTGAATGGAGAGGTATCTTTACGTTTCtaaGGATCC PmpEMREVPPSILLKPILNPYHMTGLFFPKVNLLGDTHNLTDYHLDNLKCILACLQRTPYEGAAFTVTDYLGFSD37 PmpE_EcOpt_dSig_TQKDGIFCFKNLTPESGGVIGSPTQNTPTIKIHNTIGPVLFENNTCHRLWTQTDPENEGNKAREGGAIHAGdPMPDVYISNNQNLVGFIKNFAYVQGGAISANTFAYKENKSSFLCLNNSCIQTKTGGKGGAIYVSTSCSFENNNKE. coli codonDLLFIQNSGCAGGAIFSPTCSLIGNQGDIVFYSNHGFKNVDNATNESGDGGAIKVTTRLDITNNGSQIFFSoptimized,DNISRNFGGAIHAPCLHLVGNGPTYFTNNIANHTGGAIYITGTETSKISADHHAIIFDNNISANATNADGSN-terminalSSNTNPPHRNAITMDNSAGGIELGAGKSQNLIFYDPIQVTNAGVTVDFNKDASQTGCVVFSGATVLSADISnuclearQANLQTKTPAELTLSHPAGYIPSPKHKSPLIANTLWGNIAMATENLKNSSGQELLDRPFWGITGGGLGMMVlocalizationYQEPRKDHPGFHMHTSGYSAGMITGNTHTFSLRFSQSYTKLNERYAKNYVSSKNYSCQGEMLLSLQEGLMLsignal removed,TKLIGLYSYGNHNSHHFYTQGEDLSSQGEFHSQTFGGAVFFDLPLKPFGRTHILTAPFLGAIGMYSKLSSFadhesion domainTEVGAYPRTFITETPLINVLIPIGVKGSFMNATHRPQAWTVELAYQPVLYRQEPSISTQLLAGKGMWFGHG(ChlamPMP_M) SPASRHALAYKISQKTQLLRFATLQLQYHGYYSSSTFCNYLNGEVSLRF removedPmpFATGACTCGCAGAATTCTCCCTCTTTCACTTGTTTTCATTCCTTTATCTTGTATTTCGGCCAGTGAAACCGA38 TC_RS01330-TACACTCAAACTTCCGAACTTGACTTTTGGTGGTAGAGAGATTGAATTCATAGTTACTCCGCCTAGCTCCA124632 (reversed)TTGCTGCTCAATACATCACTTACGCAAATGTTTCTAATTATAGAGGGAACTTTACTATTTCAAGTTGTACGOriginal databaseCAGGATCAATGGTTTTCGAGAGGTTTAAGCACTACAAACTCTAGTGGAGCTTTTGTTGAGTCTATGACTTCsequenceTTTCACAGCCATTGACAATGCAGACTTGTTTTTTTGTAACAATTATTGCACCCATCAGGGAGGAGGGGGAGCTATAAATGCTACAGGCCTTATTAGCTTTAAAAACAACCAAAACATATTGTTCTATAATAATACAACTATTGGAACTCAATTTACAGGAGTAGCATTAAGAACCGAAAGGAATCGCGGAGGGGCTTTATACGGATCAAGCATCGAGCTAATTAATAATCATAGCTTAAATTTTATCAATAACACTTCTGGGGATATGGGAGGAGCCGTATCCACAATCCAAAACCTAGTTATCAAAAATACGTCCGGAATAGTTGCTTTTGAAAATAACCATACTACTGATCACATACCCAACACATTTGCTACAATTCTTGCTCGAGGAGGAGCTGTTGGCTGCCAAGGTGCCTGCGAAATCTCACACAATACTGGTCCGGTAGTCTTCAATTCCAACTATGGAGGATACGGAGGAGCTATCAGCACCGGGGGACAGTGTATTTTTAGAGATAATAAGGATAAGCTTATTTTTATAAATAATAGCGCTTTAGGATGGCATAACACTAGTGCTCAAGGAAATGGAGCAGTTATAAGCGCAGGAGGAGAGTTTGGTCTTCTAAATAATAAAGGCCCTATCTACTTTGAGAATAATAATGCCTCATACATAGCAGGAGCTATTTCCTGCAACAACCTTAATTTTCAAGAAAATGGTCCTATCTATTTTCTTAATAATTCGGCTCTGTATGGAGGAGCTTTTCACCTATTTGCAAGCCCAGCTGCGAACTATATTCATACTGGCTCTGGGGATATTATCTTCAACAATAATACAGAGCTTTCAACTACCGGAATGTCAGCAGGTTTGCGAAAACTTTTTTATATTCCTGGAACAACCAACAATAACCCTATCACCCTATCTCTTGGTGCTAAGAAAGATACTCGCATCTATTTTTATGATCTTTTTCAATGGGGAGGCTTAAAAAAAGCTAATACACCCCCTGAAAATAGCCCGCACACCGTTACCATCAATCCTTCGGATGAGTTCTCTGGCGCTGTTGTGTTTTCATACAAAAACATATCCAGTGATCTACAAGCTCACATGATTGCCAGTAAAACTCATAACCAAATTAAAGACTCCCCCACTACCTTGAAGTTTGGGACTATGTCCATAGAAAATGGCGCAGAGTTTGAATTTTTCAATGGCCCTCTTACTCAAGAAAGCACTAGCCTTCTTGCTTTAGGACAAGATTCTATTCTTACTGTAGGGAAAGACGCTTCTCTCACTATTACGCATCTTGGAATCATTTTGCCAGGTCTTCTCAATGACCAAGGTACTACAGCTCCACGTATTCGTGTTAATCCCCAAGATATGACACAGAATACAAACTCTAACCAAGCTCCAGTAAGCACAGAGAACGTGGCAACTCAAAAGATCTTTTTCTCCGGTCTTGTCTCGTTAGTAGATGAAAATTACGAATCAGTTTATGACAGCTGCGACCTATCCCGAGGAAAAGCAAATCAACCCATTTTACATATCGAAACGACTAATGATGCGCAGTTAAGCAATGATTGGAAAAACACTCTCAATACCTCGCTATATTCTTTACCACATTACGGATACCAAGGACTCTGGACATCTAATTGGATGACAACCACCCGTACGGTCTCTCTTACCAATAGTACAGAGACTCAAACAGCCAACAATTCTATTCAAGAACAAAAAAACACTAGCGAAACTTTTGATTCCAACAGTACAACTACAGCTAAGATTCCTTCCATTAGAGCTTCTACAGGAGGAACAACTCCTCTTGCTACAACGGACGTAACAGTCACTAGACACTCCTTAGTAGTGAGCTGGACCCCAATCGGATATATAGCAGATCCTGCTCGTAGAGGGGATCTTATTGCGAATAATTTAGTGTCTTCTGGAAGAAATACAACCCTGTACTTACGTTCATTACTACCAGATGACTCTTGGTTCGCTTTACAAGGATCTGCAGCTACGCTATTCACCAAACAGCAGAAACGCTTAGATTATCACGGATATTCTTCTGCATCGAAAGGATATGCTATATCTTCACAAGCATCAGGAGCACACGGACATAAGTTTTTATTTTCCTTTTCCCAATCCTCCGACACAATGAAAGAGAAACGTACCAATAATAAAATTTCTTCTCGTTATTATCTCTCCGCTCTGTGTTTTGAACAACCTATGTTTGATCGTATCGCTCTTATTGGAGCAGCTGCTTATAACTATGGTACTCATAAAACATATAACTTCTATGGAACGAAAAAGTTTTCTAAAGGGAACTTTCACTCTACGACTCTGGGGGGCTCTCTACGTTGCGAACTGCGGGATAGTATGCCTTTCCAATCGATTATGTTGACACCATTCATTCAAGCTCTCATCTCCCGAACAGAGCCTGCATCTATCCAGGAGCAGGGAGACCTGGCTAGATTATTTTCGTTAAAACAACCACATACAGCTGTTGTTTCTCCAATAGGAATTAAAGGTGTTTATTCTTCGAATAAATGGCCAACTGTATCCTGCGAAATGGAGGTAGCATACCAGCCTACTCTTTACTGGAAGCGCCCCATTCTTAATACCGTTTTAATCAAAAACAATGGTTCTTGGGAAACAACAAACACTCCTTTAGCTAAGCATTCCTTTTATGGGAGAGGATCATCTTCTCTAAAATTCTCTTATCTTAAACTATTCGCTAATTATCAAGCGCAGGTGGCTACTTCTACAGTCTCACACTACATGAATGCAGGAGGGGCTCTGGTCTTTTAA PmpFMTRRILPLSLVFIPLSCISASETDTLKLPNLTFGGREIEFIVTPPSSIAAQYITYANVSNYRGNFTISSCT39 PMPF_CHLMUQDQWFSRGLSTTNSSGAFVESMTSFTAIDNADLFFCNNYCTHQGGGGAINATGLISFKNNQNILFYNNTTIGTQFTGVALRTERNRGGALYGSSIELINNHSLNFINNTSGDMGGAVSTIQNLVIKNTSGIVAFENNHTTDHIPNTFATILARGGAVGCQGACEISHNTGPVVFNSNYGGYGGAISTGGQCIFRDNKDKLIFINNSALGWHNTSAQGNGAVISAGGEFGLLNNKGPIYFENNNASYIAGAISCNNLNFQENGPIYFLNNSALYGGAFHLFASPAANYIHTGSGDIIFNNNTELSTTGMSAGLRKLFYIPGTTNNNPITLSLGAKKDTRIYFYDLFQWGGLKKANTPPENSPHTVTINPSDEFSGAVVFSYKNISSDLQAHMIASKTHNQIKDSPTTLKFGTMSIENGAEFEFFNGPLTQESTSLLALGQDSILTVGKDASLTITHLGIILPGLLNDQGTTAPRIRVNPQDMTQNTNSNQAPVSTENVATQKIFFSGLVSLVDENYESVYDSCDLSRGKANQPILHIETTNDAQLSNDWKNTLNTSLYSLPHYGYQGLWTSNWMTTTRTVSLTNSTETQTANNSIQEQKNTSETFDSNSTTTAKIPSIRASTGGTTPLATTDVTVTRHSLVVSWTPIGYIADPARRGDLIANNLVSSGRNTTLYLRSLLPDDSWFALQGSAATLFTKQQKRLDYHGYSSASKGYAISSQASGAHGHKFLFSFSQSSDTMKEKRTNNKISSRYYLSALCFEQPMFDRIALIGAAAYNYGTHKTYNFYGTKKFSKGNFHSTTLGGSLRCELRDSMPFQSIMLTPFIQALISRTEPASIQEQGDLARLFSLKQPHTAVVSPIGIKGVYSSNKWPTVSCEMEVAYQPTLYWKRPILNTVLIKNNGSWETTNTPLAKHSFYGRGSSSLKFSYLKLFANYQAQVATSTVSHYMNAGGALVF PmpFCATATGAGTGAAACCGATACACTGAAACTGCCGAACTTGACTTTTGGTGGTCGCGAGATTGAATTCATTGT40 PmpF_EcOpt_dSigTACTCCGCCTAGCTCCATTGCTGCTCAATACATCACTTACGCAAATGTTTCTAATTATCGCGGGAACTTTAE. coli codonCTATTTCAAGTTGTACGCAGGATCAATGGTTTTCGCGCGGTTTAAGCACTACAAACTCTAGTGGAGCTTTToptimized,GTTGAGTCTATGACTTCTTTCACAGCCATTGACAATGCAGACTTGTTTTTTTGTAACAATTATTGCACCCAN-terminalTCAGGGAGGAGGGGGAGCTATTAATGCTACAGGCCTGATTAGCTTTAAAAACAACCAAAACATTTTGTTCTnuclearATAATAATACAACTATTGGAACTCAATTTACAGGAGTAGCATTACGCACCGAACGCAATCGCGGAGGGGCTlocalizationTTATACGGATCAAGCATCGAGCTGATTAATAATCATAGCTTAAATTTTATCAATAACACTTCTGGGGATATsignal removedGGGAGGAGCCGTATCCACAATCCAAAACCTGGTTATCAAAAATACGTCCGGAATTGTTGCTTTTGAAAATAACCATACTACTGATCACATTCCGAACACATTTGCTACAATTCTGGCTCGCGGAGGAGCTGTTGGCTGCCAAGGTGCCTGCGAAATCTCACACAATACTGGTCCGGTAGTCTTCAATTCCAACTATGGAGGATACGGAGGAGCTATCAGCACCGGGGGACAGTGTATTTTTCGCGATAATAAGGATAAGCTGATTTTTATTAATAATAGCGCTTTAGGATGGCATAACACTAGTGCTCAAGGAAATGGAGCAGTTATTAGCGCAGGAGGAGAGTTTGGTCTGCTGAATAATAAAGGCCCTATCTACTTTGAGAATAATAATGCCTCATACATTGCAGGAGCTATTTCCTGCAACAACCTGAATTTTCAAGAAAATGGTCCTATCTATTTTCTGAATAATTCGGCTCTGTATGGAGGAGCTTTTCACCTGTTTGCAAGCCCAGCTGCGAACTATATTCATACTGGCTCTGGGGATATTATCTTCAACAATAATACAGAGCTGTCAACTACCGGAATGTCAGCAGGTTTGCGCAAACTGTTTTATATTCCTGGAACAACCAACAATAACCCTATCACCCTGTCTCTGGGTGCTAAGAAAGATACTCGCATCTATTTTTATGATCTGTTTCAATGGGGAGGCTTAAAAAAAGCTAATACACCGCCTGAAAATAGCCCGCACACCGTTACCATCAATCCTTCGGATGAGTTCTCTGGCGCTGTTGTGTTTTCATACAAAAACATTTCCAGTGAGCTCCAAGCTCACATGATTGCCAGTAAAACTCATAACCAAATTAAAGACTCCCCGACTACCTTGAAGTTTGGGACTATGTCCATTGAAAATGGCGCAGAGTTTGAATTTTTCAATGGCCCTCTGACTCAAGAAAGCACTAGCCTGCTGGCTTTAGGACAAGATTCTATTCTGACTGTAGGGAAAGACGCTTCTCTGACTATTACGCATCTGGGAATCATTTTGCCAGGTCTGCTGAATGACCAAGGTACTACAGCTCCACGTATTCGTGTTAATCCGCAAGATATGACACAGAATACAAACTCTAACCAAGCTCCAGTAAGCACAGAGAACGTGGCAACTCAAAAGATCTTTTTCTCCGGTCTGGTCTCGTTAGTAGATGAAAATTACGAATCAGTTTATGACAGCTGCGACCTGTCCCGCGGAAAAGCAAATCAACCGATTTTACATATCGAAACGACTAATGATGCGCAGTTAAGCAATGATTGGAAAAACACTCTGAATACCTCGCTGTATTCTTTACCACATTACGGATACCAAGGACTGTGGACATCTAATTGGATGACAACCACCCGTACGGTCTCTCTGACCAATAGTACAGAGACTCAAACAGCCAACAATTCTATTCAAGAACAAAAAAACACTAGCGAAACTTTTGATTCCAACAGTACAACTACAGCTAAGATTCCTTCCATTCGCGCTTCTACAGGAGGAACAACTCCCATGGCTACAACGGACGTAACAGTCACTCGCCACTCCTTAGTAGTGAGCTGGACCCCAATCGGATATATTGCAGATCCTGCTCGTCGCGGGGATCTGATTGCGAATAATTTAGTGTCTTCTGGACGCAATACAACCCTGTACTTACGTTCATTACTGCCAGATGACTCTTGGTTCGCTTTACAAGGATCTGCAGCTACGCTGTTCACCAAACAGCAGAAACGCTTAGATTATCACGGATATTCTTCTGCATCGAAAGGATATGCTATTTCTTCACAAGCATCAGGAGCACACGGACATAAGTTTTTATTTTCCTTTTCCCAATCCTCCGACACAATGAAAGAGAAACGTACCAATAATAAAATTTCTTCTCGTTATTATCTGTCCGCTCTGTGTTTTGAACAACCTATGTTTGATCGTATCGCTCTGATTGGAGCAGCTGCTTATAACTATGGTACTCATAAAACATATAACTTCTATGGAACGAAAAAGTTTTCTAAAGGGAACTTTCACTCTACGACTCTGGGGGGCTCTCTGCGTTGCGAACTGCGGGATAGTATGCCTTTCCAATCGATTATGTTGACACCATTCATTCAAGCTCTGATCTCCCGCACAGAGCCTGCATCTATCCAGGAGCAGGGAGACCTGGCTCGCTTATTTTCGTTAAAACAACCACATACAGCTGTTGTTTCTCCAATTGGAATTAAAGGTGTTTATTCTTCGAATAAATGGCCAACTGTATCCTGCGAAATGGAGGTAGCATACCAGCCTACTCTGTACTGGAAGCGCCCGATTCTGAATACCGTTTTAATCAAAAACAATGGTTCTTGGGAAACAACAAACACTCCTTTAGCTAAGCATTCCTTTTATGGGCGCGGATCATCTTCTCTGAAATTCTCTTATCTGAAACTGTTCGCTAATTATCAAGCGCAGGTGGCTACTTCTACAGTCTCACACTACATGAATGCAGGAGGGGCTCTGGTCTTTtaaGGATCC PmpFMSETDTLKLPNLTFGGREIEFIVTPPSSIAAQYITYANVSNYRGNFTISSCTQDQWFSRGLSTTNSSGAFV41 PmpF_EcOpt_dSigESMTSFTAIDNADLFFCNNYCTHQGGGGAINATGLISFKNNQNILFYNNTTIGTQFTGVALRTERNRGGALE. coli codonYGSSIELINNHSLNFINNTSGDMGGAVSTIQNLVIKNTSGIVAFENNHTTDHIPNTFATILARGGAVGCQGoptimized,ACEISHNTGPVVFNSNYGGYGGAISTGGQCIFRDNKDKLIFINNSALGWHNTSAQGNGAVISAGGEFGLLNN-terminalNKGPIYFENNNASYIAGAISCNNLNFQENGPIYFLNNSALYGGAFHLFASPAANYIHTGSGDIIFNNNTELnuclearSTTGMSAGLRKLFYIPGTTNNNPITLSLGAKKDTRIYFYDLFQWGGLKKANTPPENSPHTVTINPSDEFSGlocalizationAVVFSYKNISSELQAHMIASKTHNQIKDSPTTLKFGTMSIENGAEFEFFNGPLTQESTSLLALGQDSILTVsignal removedGKDASLTITHLGIILPGLLNDQGTTAPRIRVNPQDMTQNTNSNQAPVSTENVATQKIFFSGLVSLVDENYESVYDSCDLSRGKANQPILHIETTNDAQLSNDWKNTLNTSLYSLPHYGYQGLWTSNWMTTTRTVSLTNSTETQTANNSIQEQKNTSETFDSNSTTTAKIPSIRASTGGTTPMATTDVTVTRHSLVVSWTPIGYIADPARRGDLIANNLVSSGRNTTLYLRSLLPDDSWFALQGSAATLFTKQQKRLDYHGYSSASKGYAISSQASGAHGHKFLFSFSQSSDTMKEKRTNNKISSRYYLSALCFEQPMFDRIALIGAAAYNYGTHKTYNFYGTKKFSKGNFHSTTLGGSLRCELRDSMPFQSIMLTPFIQALISRTEPASIQEQGDLARLFSLKQPHTAVVSPIGIKGVYSSNKWPTVSCEMEVAYQPTLYWKRPILNTVLIKNNGSWETTNTPLAKHSFYGRGSSSLKFSYLKLFANYQAQVATSTVSHYMNAGGALVF PmpFCATATGAGTGAAACCGATACACTGAAACTGCCGAACTTGACTTTTGGTGGTCGCGAGATTGAATTCATTGT42 PmpF_EcOpt_dSig_TACTCCGCCTAGCTCCATTGCTGCTCAATACATCACTTACGCAAATGTTTCTAATTATCGCGGGAACTTTAdPMPCTATTTCAAGTTGTACGCAGGATCAATGGTTTTCGCGCGGTTTAAGCACTACAAACTCTAGTGGAGCTTTTE. coli codonGTTGAGTCTATGACTTCTTTCACAGCCATTGACAATGCAGACTTGTTTTTTTGTAACAATTATTGCACCCAoptimized,TCAGGGAGGAGGGGGAGCTATTAATGCTACAGGCCTGATTAGCTTTAAAAACAACCAAAACATTTTGTTCTN-terminalATAATAATACAACTATTGGAACTCAATTTACAGGAGTAGCATTACGCACCGAACGCAATCGCGGAGGGGCTnuclearTTATACGGATCAAGCATCGAGCTGATTAATAATCATAGCTTAAATTTTATCAATAACACTTCTGGGGATATlocalizationGGGAGGAGCCGTATCCACAATCCAAAACCTGGTTATCAAAAATACGTCCGGAATTGTTGCTTTTGAAAATAsignal removed,ACCATACTACTGATCACATTCCGAACACATTTGCTACAATTCTGGCTCGCGGAGGAGCTGTTGGCTGCCAAadhesion domainGGTGCCTGCGAAATCTCACACAATACTGGTCCGGTAGTCTTCAATTCCAACTATGGAGGATACGGAGGAGC(ChlamPMP_M)TATCAGCACCGGGGGACAGTGTATTTTTCGCGATAATAAGGATAAGCTGATTTTTATTAATAATAGCGCTTremovedTAGGATGGCATAACACTAGTGCTCAAGGAAATGGAGCAGTTATTAGCGCAGGAGGAGAGTTTGGTCTGCTGAATAATAAAGGCCCTATCTACTTTGAGAATAATAATGCCTCATACATTGCAGGAGCTATTTCCTGCAACAACCTGAATTTTCAAGAAAATGGTCCTATCTATTTTCTGAATAATTCGGCTCTGTATGGAGGAGCTTTTCACCTGTTTGCAAGCCCAGCTGCGAACTATATTCATACTGGCTCTGGGGATATTATCTTCAACAATAATACAGAGCTGTCAACTACCGGAATGTCAGCAGGTTTGCGCAAACTGTTTTATATTCCTGGAACAACCAACAATAACCCTATCACCCTGTCTCTGGGTGCTAAGAAAGATACTCGCATCTATTTTTATGATCTGTTTCAATGGGGAGGCTTAAAAAAAGCTAATACACCGCCTGAAAATAGCCCGCACACCGTTACCATCAATCCTTCGGATGAGTTCTCTGGCGCTGTTGTGTTTTCATACAAAAACATTTCCAGTGAGCTCCAAGCTCACATGATTGCCAGTAAAACTCATAACCAAATTAAAGACTCCCCGACTACCTTGAAGTTTAATTCTATTCAAGAACAAAAAAACACTAGCGAAACTTTTGATTCCAACAGTACAACTACAGCTAAGATTCCTTCCATTCGCGCTTCTACAGGAGGAACAACTCCCATGGCTACAACGGACGTAACAGTCACTCGCCACTCCTTAGTAGTGAGCTGGACCCCAATCGGATATATTGCAGATCCTGCTCGTCGCGGGGATCTGATTGCGAATAATTTAGTGTCTTCTGGACGCAATACAACCCTGTACTTACGTTCATTACTGCCAGATGACTCTTGGTTCGCTTTACAAGGATCTGCAGCTACGCTGTTCACCAAACAGCAGAAACGCTTAGATTATCACGGATATTCTTCTGCATCGAAAGGATATGCTATTTCTTCACAAGCATCAGGAGCACACGGACATAAGTTTTTATTTTCCTTTTCCCAATCCTCCGACACAATGAAAGAGAAACGTACCAATAATAAAATTTCTTCTCGTTATTATCTGTCCGCTCTGTGTTTTGAACAACCTATGTTTGATCGTATCGCTCTGATTGGAGCAGCTGCTTATAACTATGGTACTCATAAAACATATAACTTCTATGGAACGAAAAAGTTTTCTAAAGGGAACTTTCACTCTACGACTCTGGGGGGCTCTCTGCGTTGCGAACTGCGGGATAGTATGCCTTTCCAATCGATTATGTTGACACCATTCATTCAAGCTCTGATCTCCCGCACAGAGCCTGCATCTATCCAGGAGCAGGGAGACCTGGCTCGCTTATTTTCGTTAAAACAACCACATACAGCTGTTGTTTCTCCAATTGGAATTAAAGGTGTTTATTCTTCGAATAAATGGCCAACTGTATCCTGCGAAATGGAGGTAGCATACCAGCCTACTCTGTACTGGAAGCGCCCGATTCTGAATACCGTTTTAATCAAAAACAATGGTTCTTGGGAAACAACAAACACTCCTTTAGCTAAGCATTCCTTTTATGGGCGCGGATCATCTTCTCTGAAATTCTCTTATCTGAAACTGTTCGCTAATTATCAAGCGCAGGTGGCTACTTCTACAGTCTCACACTACATGAATGCAGGAGGGGCTCTGGTCTTTtaaGGATCCPmpGATGCAAACGCCTTTTCATAAGTTCTTTCTTCTAGCAATGCTATCTTACTCTTTATTGCAAGGAGGGCATGC43 TC_RS01335-GGCAGATATTTCCATGCCTCCGGGAATTTATGATGGGACAACATTGACGGCGCCATTTCCCTACACTGTGA124633 OriginalTCGGAGATCCCAGAGGGACAAAGGTTACTTCATCGGGATCGCTAGAGTTGAAAAACCTGGACAATTCCATTdatabase sequenceGCGACTTTACCTCTAAGTTGTTTTGGTAATTTGTTGGGGAATTTCACTATTGCAGGAAGAGGGCATTCGTTAGTATTTGAGAATATACGAACATCTACAAATGGGGCGGCATTGAGTAATCATGCTCCTTCTGGACTGTTTGTAATTGAAGCTTTTGATGAACTCTCTCTTTTGAATTGTAATTCATTGGTATCTGTAGTTCCTCAAACAGGGGGTACGACTACTTCTGTTCCTTCTAATGGGACGATCTATTCTAGAACAGATCTTGTTCTAAGAGATATCAAGAAGGTTTCTTTCTATAGTAACTTAGTTTCTGGAGATGGGGGAGCTATAGATGCACAAAGTTTAATGGTTAACGGAATTGAAAAACTTTGTACCTTCCAAGAAAATGTAGCGCAGTCCGATGGGGGAGCGTGTCAGGTAACAAAGACCTTCTCTGCTGTGGGCAATAAGGTTCCTTTGTCTTTTTTAGGCAATGTTGCTGGTAATAAGGGGGGAGGAGTTGCTGCTGTCAAAGATGGTCAGGGGGCAGGAGGGGCGACTGATCTATCGGTTAATTTTGCCAATAATACTGCTGTAGAATTTGAGGGAAATAGTGCTCGAATAGGTGGAGGGATCTACTCGGACGGAAATATTTCCTTTTTAGGGAATGCAAAGACAGTTTTCCTAAGTAACGTAGCTTCGCCTATTTATGTTGACCCTGCTGCTGCAGGAGGACAGCCCCCTGCAGATAAAGATAACTATGGAGATGGAGGAGCCATCTTCTGCAAAAATGATACTAACATAGGTGAAGTCTCTTTCAAAGACGAGGGTGTTGTTTTCTTTAGTAAAAATATTGCCGCAGGAAAGGGGGGCGCTATTTATGCTAAGAAACTGACAATTTCTGACTGTGGTCCGGTCCAGTTTCTTGGTAATGTCGCGAATGACGGGGGCGCTATTTATCTAGTAGATCAGGGGGAACTTAGTCTATCTGCTGATCGCGGAGATATTATTTTTGATGGAAATTTAAAGAGAATGGCTACGCAAGGCGCTGCCACCGTCCATGATGTAATGGTTGCATCGAATGCTATCTCTATGGCTACAGGGGGGCAAATCACAACATTAAGGGCTAAGGAAGGTCGCCGAATTCTTTTTAATGACCCTATTGAAATGGCGAATGGACAACCTGTAATACAAACTCTTACAGTAAACGAGGGCGAAGGATATACGGGGGACATTGTTTTTGCTAAAGGTGATAATGTTTTGTACTCAAGTATTGAGCTGAGTCAGGGAAGAATTATTCTCCGAGAGCAAACAAAATTATTGGTTAACTCCCTGACTCAGACTGGAGGGAGTGTACATATGGAAGGGGGGAGTACACTAGACTTTGCAGTAACAACGCCACCAGCTGCTAATTCGATGGCTCTTACTAATGTACACTTCTCCTTAGCTTCTTTACTAAAAAATAATGGGGTTACAAATCCTCCAACGAATCCTCCAGTACAGGTTTCTAGTCCAGCTGTAATTGGTAATACAGCTGCTGGTACTGTTACGATTTCTGGTCCGATCTTTTTTGAAGATTTAGATGAAACTGCTTACGATAATAATCAGTGGTTAGGTGCGGATCAAACTATTGATGTGCTGCAGTTGCATTTAGGAGCGAATCCTCCGGCTAACGCTCCAACTGATTTGACTTTAGGGAACGAAAGTTCTAAATATGGGTATCAAGGAAGTTGGACACTTCAATGGGAACCAGATCCTGCGAATCCTCCACAGAACAATAGCTACATGTTGAAGGCAAGCTGGACTAAAACAGGTTATAATCCTGGTCCGGAGCGCGTAGCTTCTCTGGTCTCTAATAGTCTTTGGGGATCCATTTTAGATGTGCGTTCCGCGCATTCTGCGATTCAAGCAAGTATAGATGGACGAGCTTATTGTCGGGGTATTTGGATTTCTGGGATTTCGAACTTTTTCTATCATGATCAGGATGCTTTAGGACAGGGGTATCGTCATATTAGTGGGGGATATTCGATAGGAGCAAACTCTTATTTCGGGTCTTCTATGTTTGGACTTGCTTTTACTGAAACTTTTGGTAGGTCCAAAGATTATGTGGTCTGTCGATCTAACGATCACACTTGTGTAGGCTCTGTTTACTTATCCACTAGACAAGCGTTATGCGGATCCTGTTTATTTGGAGATGCTTTTGTTCGGGCGAGTTACGGATTTGGAAATCAGCATATGAAGACCTCTTATACATTTGCTGAAGAGAGTAATGTGCGTTGGGATAATAACTGTGTAGTGGGAGAAGTTGGAGCTGGGCTCCCTATCATGCTCGCTGCATCTAAGCTTTATCTAAATGAGTTGCGTCCCTTCGTGCAAGCAGAGTTTGCTTATGCAGAGCATGAATCTTTTACAGAGAGAGGGGATCAGGCTAGGGAGTTTAAGAGTGGGCATCTTATGAATCTATCTATTCCAGTTGGGGTGAAGTTTGATCGATGCTCTAGTAAACATCCTAACAAGTATAGTTTTATGGGAGCTTATATCTGTGATGCTTACCGGTCCATTTCTGGAACGGAGACAACACTCCTGTCTCATAAAGAGACTTGGACAACAGATGCTTTCCATTTAGCAAGGCATGGAGTTATGGTCAGAGGATCTATGTATGCTTCTTTAACAGGTAATATAGAAGTCTATGGCCATGGAAAATATGAATACAGGGATGCCTCTCGAGGGTATGGTTTAAGTATTGGAAGTAAAATCCGATTCTAA PmpGMMQTPFHKETLLAMLSYSLLQGGHAADISMPPGIYDGTTLTAPFPYTVIGDPRGTKVTSSGSLELKNLDNS44 PmpG_CHLMUIATLPLSCFGNLLGNFTIAGRGHSLVFENIRTSTNGAALSNHAPSGLFVIEAFDELSLLNCNSLVSVVPQTGGTTTSVPSNGTIYSRTDLVLRDIKKVSFYSNLVSGDGGAIDAQSLMVNGIEKLCTFQENVAQSDGGACQVTKTFSAVGNKVPLSFLGNVAGNKGGGVAAVKDGQGAGGATDLSVNFANNTAVEFEGNSARIGGGIYSDGNISFLGNAKTVFLSNVASPIYVDPAAAGGQPPADKDNYGDGGAIFCKNDTNIGEVSFKDEGVVFFSKNIAAGKGGAIYAKKLTISDCGPVQFLGNVANDGGAIYLVDQGELSLSADRGDIIFDGNLKRMATQGAATVHDVMVASNAISMATGGQITTLRAKEGRRILFNDPIEMANGQPVIQTLTVNEGEGYTGDIVFAKGDNVLYSSIELSQGRIILREQTKLLVNSLTQTGGSVHMEGGSTLDFAVTTPPAANSMALTNVHFSLASLLKNNGVTNPPTNPPVQVSSPAVIGNTAAGTVTISGPIFFEDLDETAYDNNQWLGADQTIDVLQLHLGANPPANAPTDLTLGNESSKYGYQGSWTLQWEPDPANPPQNNSYMLKASWTKTGYNPGPERVASLVSNSLWGSILDVRSAHSAIQASIDGRAYCRGIWISGISNFFYHDQDALGQGYRHISGGYSIGANSYFGSSMFGLAFTETFGRSKDYVVCRSNDHTCVGSVYLSTRQALCGSCLFGDAFVRASYGFGNQHMKTSYTFAEESNVRWDNNCVVGEVGAGLPIMLAASKLYLNELRPFVQAEFAYAEHESFTERGDQAREFKSGHLMNLSIPVGVKFDRCSSKHPNKYSFMGAYICDAYRSISGTETTLLSHKETWTTDAFHLARHGVMVRGSMYASLTGNIEVYGHGKYEYRDASRGYGLSIGSKIRF PmpGCATATGGCAGATATTTCCATGCCTCCGGGAATTTATGATGGGACAACATTGACGGCGCCATTTCCGTACAC45 PmpG_EcOpt_dSigTGTGATCGGAGATCCGCGCGGGACAAAGGTTACTTCATCGGGATCGCTGGAGTTGAAAAACCTGGACAATTE. coli codonCCATTGCGACTTTACCTCTGAGTTGTTTTGGTAATTTGTTGGGGAATTTCACTATTGCAGGACGCGGGCAToptimized,TCGTTAGTATTTGAGAATATTCGCACATCTACAAATGGGGCGGCATTGAGTAATCATGCTCCTTCTGGACTN-terminalGTTTGTAATTGAAGCTTTTGATGAACTGTCTCTGTTGAATTGTAATTCATTGGTATCTGTAGTTCCTCAAAnuclearCAGGGGGTACGACTACTTCTGTTCCTTCTAATGGGACGATCTATTCCCGCACAGATCTGGTTCTGCGCGATlocalizationATCAAGAAGGTTTCTTTCTATAGTAACTTAGTTTCTGGAGATGGGGGAGCTATTGATGCACAAAGTTTAATsignal removedGGTTAACGGAATTGAAAAACTGTGTACCTTCCAAGAAAATGTAGCGCAGTCCGATGGGGGAGCGTGTCAGGTAACAAAGACCTTCTCTGCTGTGGGCAATAAGGTTCCTTTGTCTTTTTTAGGCAATGTTGCTGGTAATAAGGGGGGAGGAGTTGCTGCTGTCAAAGATGGTCAGGGGGCAGGAGGGGCGACTGATCTGTCGGTTAATTTTGCCAATAATACTGCTGTAGAATTTGAGGGAAATAGTGCTCGCATTGGTGGAGGGATCTACTCGGACGGAAATATTTCCTTTTTAGGGAATGCAAAGACAGTTTTCCTGAGTAACGTAGCTTCGCCTATTTATGTTGACCCTGCTGCTGCAGGAGGACAGCCGCCTGCAGATAAAGATAACTATGGAGATGGAGGAGCCATCTTCTGCAAAAATGATACTAACATTGGTGAAGTCTCTTTCAAAGACGAGGGTGTTGTTTTCTTTAGTAAAAATATTGCCGCAGGAAAGGGGGGCGCTATTTATGCTAAGAAACTGACAATTTCTGACTGTGGTCCGGTCCAGTTTCTGGGTAATGTCGCGAATGACGGGGGCGCTATTTATCTGGTAGATCAGGGGGAACTGAGTCTGTCTGCTGATCGCGGAGATATTATTTTTGATGGAAATTTAAAGCGCATGGCTACGCAAGGCGCTGCCACCGTCCATGATGTAATGGTTGCATCGAATGCTATCTCTATGGCTACAGGGGGGCAAATCACAACATTACGCGCTAAGGAAGGTCGCCGCATTCTGTTTAATGACCCTATTGAAATGGCGAATGGACAACCTGTAATTCAAACTCTGACAGTAAACGAGGGCGAAGGATATACGGGGGACATTGTTTTTGCTAAAGGTGATAATGTTTTGTACTCAAGTATTGAGCTCAGTCAGGGACGCATTATTCTGCGCGAGCAAACAAAATTATTGGTTAACTCCCTGACTCAGACTGGAGGGAGTGTACACATGGAAGGGGGGAGTACACTGGACTTTGCAGTAACAACGCCACCAGCTGCTAATTCGATGGCTCTGACTAATGTACACTTCTCCTTAGCTTCTTTACTGAAAAATAATGGGGTTACAAATCCTCCAACGAATCCTCCAGTACAGGTTTCTAGTCCAGCTGTAATTGGTAATACAGCTGCTGGTACTGTTACGATTTCTGGTCCGATCTTTTTTGAAGATTTAGATGAAACTGCTTACGATAATAATCAGTGGTTAGGTGCGGATCAAACTATTGATGTGCTGCAGTTGCATTTAGGAGCGAATCCTCCGGCTAACGCTCCAACTGATTTGACTTTAGGGAACGAAAGTTCTAAATATGGGTATCAAGGAAGTTGGACACTGCAATGGGAACCAGATCCTGCGAATCCTCCACAGAACAATAGCTACATGTTGAAGGCAAGCTGGACTAAAACAGGTTATAATCCTGGTCCGGAGCGCGTAGCTTCTCTGGTCTCTAATAGTCCCATGGGTTCCATTTTAGATGTGCGTTCCGCGCATTCTGCGATTCAAGCAAGTATTGATGGACGCGCTTATTGTCGGGGTATTTGGATTTCTGGGATTTCGAACTTTTTCTATCATGATCAGGATGCTTTAGGACAGGGGTATCGTCATATTAGTGGGGGATATTCGATTGGAGCAAACTCTTATTTCGGGTCTTCTATGTTTGGACTGGCTTTTACTGAAACTTTTGGTCGCTCCAAAGATTATGTGGTCTGTCGCTCTAACGATCACACTTGTGTAGGCTCTGTTTACTTATCCACTCGCCAAGCGTTATGCGGGTCCTGTTTATTTGGAGATGCTTTTGTTCGGGCGAGTTACGGATTTGGAAATCAGCACATGAAGACCTCTTATACATTTGCTGAAGAGAGTAATGTGCGTTGGGATAATAACTGTGTAGTGGGAGAAGTTGGAGCTGGGCTGCCTATCATGCTGGCTGCATCTAAGCTGTATCTGAATGAGTTGCGTCCGTTCGTGCAAGCAGAGTTTGCTTATGCAGAGCATGAATCTTTTACAGAGCGCGGGGATCAGGCTCGCGAGTTTAAGAGTGGGCATCTGATGAATCTGTCTATTCCAGTTGGGGTGAAGTTTGATCGCTGCTCTAGTAAACATCCTAACAAGTATAGTTTTATGGGAGCTTATATCTGTGATGCTTACCGGTCCATTTCTGGAACGGAGACAACACTGCTGTCTCATAAAGAGACTTGGACAACAGATGCTTTCCATTTAGCACGTCATGGAGTTATGGTCCGCGGATCTATGTATGCTTCTTTAACAGGTAATATTGAAGTCTATGGGCATGGAAAATATGAATACCGCGATGCCTCTCGCGGGTATGGTTTAAGTATTGGAAGTAAAATCCGCTTCtaaGGATCC PmpGMADISMPPGIYDGTTLTAPFPYTVIGDPRGTKVTSSGSLELKNLDNSIATLPLSCFGNLLGNFTIAGRGHS46 PmpG_EcOpt_dSigLVFENIRTSTNGAALSNHAPSGLFVIEAFDELSLLNCNSLVSVVPQTGGTTTSVPSNGTIYSRTDLVLRDIE. coli codonKKVSFYSNLVSGDGGAIDAQSLMVNGIEKLCTFQENVAQSDGGACQVTKTFSAVGNKVPLSFLGNVAGNKGoptimized,GGVAAVKDGQGAGGATDLSVNFANNTAVEFEGNSARIGGGIYSDGNISFLGNAKTVFLSNVASPIYVDPAAN-terminalAGGQPPADKDNYGDGGAIFCKNDTNIGEVSFKDEGVVFFSKNIAAGKGGAIYAKKLTISDCGPVQFLGNVAnuclearNDGGAIYLVDQGELSLSADRGDIIFDGNLKRMATQGAATVHDVMVASNAISMATGGQITTLRAKEGRRILFlocalizationNDPIEMANGQPVIQTLTVNEGEGYTGDIVFAKGDNVLYSSIELSQGRIILREQTKLLVNSLTQTGGSVHMEsignal removedGGSTLDFAVTTPPAANSMALTNVHFSLASLLKNNGVTNPPTNPPVQVSSPAVIGNTAAGTVTISGPIFFEDLDETAYDNNQWLGADQTIDVLQLHLGANPPANAPTDLTLGNESSKYGYQGSWTLQWEPDPANPPQNNSYMLKASWTKTGYNPGPERVASLVSNSPMGSILDVRSAHSAIQASIDGRAYCRGIWISGISNFFYHDQDALGQGYRHISGGYSIGANSYFGSSMFGLAFTETFGRSKDYVVCRSNDHTCVGSVYLSTRQALCGSCLFGDAFVRASYGFGNQHMKTSYTFAEESNVRWDNNCVVGEVGAGLPIMLAASKLYLNELRPFVQAEFAYAEHESFTERGDQAREFKSGHLMNLSIPVGVKFDRCSSKHPNKYSFMGAYICDAYRSISGTETTLLSHKETWTTDAFHLARHGVMVRGSMYASLTGNIEVYGHGKYEYRDASRGYGLSIGSKIRF PmpGCATATGGCAGATATTTCCATGCCTCCGGGAATTTATGATGGGACAACATTGACGGCGCCATTTCCGTACAC47 PmpG_EcOpt_dSig_TGTGATCGGAGATCCGCGCGGGACAAAGGTTACTTCATCGGGATCGCTGGAGTTGAAAAACCTGGACAATTdPMPCCATTGCGACTTTACCTCTGAGTTGTTTTGGTAATTTGTTGGGGAATTTCACTATTGCAGGACGCGGGCATE. coli codonTCGTTAGTATTTGAGAATATTCGCACATCTACAAATGGGGCGGCATTGAGTAATCATGCTCCTTCTGGACToptimized,GTTTGTAATTGAAGCTTTTGATGAACTGTCTCTGTTGAATTGTAATTCATTGGTATCTGTAGTTCCTCAAAN-terminalCAGGGGGTACGACTACTTCTGTTCCTTCTAATGGGACGATCTATTCCCGCACAGATCTGGTTCTGCGCGATnuclearATCAAGAAGGTTTCTTTCTATAGTAACTTAGTTTCTGGAGATGGGGGAGCTATTGATGCACAAAGTTTAATlocalizationGGTTAACGGAATTGAAAAACTGTGTACCTTCCAAGAAAATGTAGCGCAGTCCGATGGGGGAGCGTGTCAGGsignal removed,TAACAAAGACCTTCTCTGCTGTGGGCAATAAGGTTCCTTTGTCTTTTTTAGGCAATGTTGCTGGTAATAAGadhesion domainGGGGGAGGAGTTGCTGCTGTCAAAGATGGTCAGGGGGCAGGAGGGGCGACTGATCTGTCGGTTAATTTTGC(ChlamPMP_M)CAATAATACTGCTGTAGAATTTGAGGGAAATAGTGCTCGCATTGGTGGAGGGATCTACTCGGACGGAAATAremovedTTTCCTTTTTAGGGAATGCAAAGACAGTTTTCCTGAGTAACGTAGCTTCGCCTATTTATGTTGACCCTGCTGCTGCAGGAGGACAGCCGCCTGCAGATAAAGATAACTATGGAGATGGAGGAGCCATCTTCTGCAAAAATGATACTAACATTGGTGAAGTCTCTTTCAAAGACGAGGGTGTTGTTTTCTTTAGTAAAAATATTGCCGCAGGAAAGGGGGGCGCTATTTATGCTAAGAAACTGACAATTTCTGACTGTGGTCCGGTCCAGTTTCTGGGTAATGTCGCGAATGACGGGGGCGCTATTTATCTGGTAGATCAGGGGGAACTGAGTCTGTCTGCTGATCGCGGAGATATTATTTTTGATGGAAATTTAAAGCGCATGGCTACGCAAGGCGCTGCCACCGTCCATGATGTAATGGTTGCATCGAATGCTATCTCTATGGCTACAGGGGGGCAAATCACAACATTACGCGCTAAGGAAGGTCGCCGCATTCTGTTTAATGACCCTATTGAAATGGCGAATGGACAACCTGTAATTCAAACTCTGACAGTAAACGAGGGCGAAGGATATACGGGGGACATTGTTTTTGCTAAAGGTGATAATGTTTTGTACTCAAGTATTGAGCTCAGTCAGACAGGTTATAATCCTGGTCCGGAGCGCGTAGCTTCTCTGGTCTCTAATAGTCCCATGGGTTCCATTTTAGATGTGCGTTCCGCGCATTCTGCGATTCAAGCAAGTATTGATGGACGCGCTTATTGTCGGGGTATTTGGATTTCTGGGATTTCGAACTTTTTCTATCATGATCAGGATGCTTTAGGACAGGGGTATCGTCATATTAGTGGGGGATATTCGATTGGAGCAAACTCTTATTTCGGGTCTTCTATGTTTGGACTGGCTTTTACTGAAACTTTTGGTCGCTCCAAAGATTATGTGGTCTGTCGCTCTAACGATCACACTTGTGTAGGCTCTGTTTACTTATCCACTCGCCAAGCGTTATGCGGGTCCTGTTTATTTGGAGATGCTTTTGTTCGGGCGAGTTACGGATTTGGAAATCAGCACATGAAGACCTCTTATACATTTGCTGAAGAGAGTAATGTGCGTTGGGATAATAACTGTGTAGTGGGAGAAGTTGGAGCTGGGCTGCCTATCATGCTGGCTGCATCTAAGCTGTATCTGAATGAGTTGCGTCCGTTCGTGCAAGCAGAGTTTGCTTATGCAGAGCATGAATCTTTTACAGAGCGCGGGGATCAGGCTCGCGAGTTTAAGAGTGGGCATCTGATGAATCTGTCTATTCCAGTTGGGGTGAAGTTTGATCGCTGCTCTAGTAAACATCCTAACAAGTATAGTTTTATGGGAGCTTATATCTGTGATGCTTACCGGTCCATTTCTGGAACGGAGACAACACTGCTGTCTCATAAAGAGACTTGGACAACAGATGCTTTCCATTTAGCACGTCATGGAGTTATGGTCCGCGGATCTATGTATGCTTCTTTAACAGGTAATATTGAAGTCTATGGGCATGGAAAATATGAATACCGCGATGCCTCTCGCGGGTATGGTTTAAGTATTGGAAGTAAAATCCGCTTCtaaGGATCC PmpGMADISMPPGIYDGTTLTAPFPYTVIGDPRGTKVTSSGSLELKNLDNSIATLPLSCFGNLLGNFTIAGRGHS48 PmpG_EcOpt_dSig_LVFENIRTSTNGAALSNHAPSGLFVIEAFDELSLLNCNSLVSVVPQTGGTTTSVPSNGTIYSRTDLVLRDIdPMPKKVSFYSNLVSGDGGAIDAQSLMVNGIEKLCTFQENVAQSDGGACQVTKTFSAVGNKVPLSFLGNVAGNKGE. coli codonGGVAAVKDGQGAGGATDLSVNFANNTAVEFEGNSARIGGGIYSDGNISFLGNAKTVFLSNVASPIYVDPAAoptimized,AGGQPPADKDNYGDGGAIFCKNDTNIGEVSFKDEGVVFFSKNIAAGKGGAIYAKKLTISDCGPVQFLGNVAN-terminalNDGGAIYLVDQGELSLSADRGDIIFDGNLKRMATQGAATVHDVMVASNAISMATGGQITTLRAKEGRRILFnuclearNDPIEMANGQPVIQTLTVNEGEGYTGDIVFAKGDNVLYSSIELSQTGYNPGPERVASLVSNSPMGSILDVRlocalizationSAHSAIQASIDGRAYCRGIWISGISNFFYHDQDALGQGYRHISGGYSIGANSYFGSSMFGLAFTETFGRSKsignal removed,DYVVCRSNDHTCVGSVYLSTRQALCGSCLFGDAFVRASYGFGNQHMKTSYTFAEESNVRWDNNCVVGEVGAadhesion domainGLPIMLAASKLYLNELRPFVQAEFAYAEHESFTERGDQAREFKSGHLMNLSIPVGVKFDRCSSKHPNKYSF(ChlamPMP_M)MGAYICDAYRSISGTETTLLSHKETWTTDAFHLARHGVMVRGSMYASLTGNIEVYGHGKYEYRDASRGYGLremoved SIGSKIRF PmpHATGCCTTTTTCTTTGAGATCTACATCATTTTGTTTTTTAGCCTGTTTATGTTCTTATTCATATGGATTAGC49 TC_RS01340-GAGTTCTCCTCAGGTACTGACCCCCAATGTAATCATCCCTTTTAAAGGAGACGATATCTATTTAAATGGGG124634 OriginalATTGCGTTTTTGCAAGTATCTATGCAGGAGCAGAGCAGGGATCGATTATTTCTGCTAATGGGCAAAATCTAdatabase sequenceACAATCGTAGGACAAAACCACACTTTATCATTTACGGATTCCCAAGGGCCAGCCCTTCAAAATTGTGCTTTCATTTCAGCAGAAGAAAAGATCTCTCTAAGAGATTTTTCGAGCCTTTTGTTTTCGAAAAATGTTTCTTGCGGGGAGAAAGGAATGATTTCAGGGAAAACCGTAAGCATTTCAGGGGGAGATAGTATAGTTTTTAAGGATAACTCTGTTGGTTATTCTTCATTACCCTCTGTGGGGCAAACTCCTACAACTCCAATTGTTGGCGATGTTTTAAAGGGATCCATTTTTTGTGTGGAGACAGGTTTAGAGATTTCTGGAGTCAAAAAAGAGCTTGTTTTCGATAACACTGCTGGGAATTTTGGGGCAGTATTCTGTAGTCGTGCCGCTCAAGGAGACACGACTTTCACAGTGAAAGACTGTAAGGGTAAAATTCTTTTTCAAGATAACGTAGGCTCTTGTGGAGGCGGCGTAATTTATAAAGGGGAAGTACTTTTCCAAGATAATGAAGGAGAAATGCTTTTCCGAGGAAATTCAGCTCATGATGATTTGGGAATTCTCGATGCTAACCCACAGCCTCCTACTGAAGTAGGAGGTGGGGGTGGTGTCATTTGTACCCCAGAGAAAACGGTAACTTTTAAGGGGAATAAAGGGCCTATTACCTTTGATTATAATTTTGCAAAAGGTCGAGGAGGGGCAATCCAATCACAGACCTTTTCTTTGGTAGCTGATAGTGCTGTTGTTTTCAGTAATAATACAGCTGAGAAAGGTGGAGGCGCCATTTATGCTCTTGAGGTTAACGTGAGCACAAATGGAGGATCTATTCTTTTTGAGGGAAATAGAGCTTCTGAGGGTGGGGCTATCTGTGTGAGCGAGCCGATCGCTGCTAATAATGGAGGGCTCACTTTACATGCTGCTGATGGGGACATTATTTTCTCGAAAAATATGACGAGTGATCGTCCTGGAGAACGCAGTGCAATCCGGATCTTAGATAGTGGAACAAATGTCTCTTTAAATGCTTCAGGGGCATCGAAGATGATTTTTTATGATCCTGTTGTGCAAAATAATCCCGCAACTCCACCTACTGGTACGTCTGGGGAAATTAAGATCAATGAGTCCGGGAGTGGATCGGTTGTGTTTACAGCAGAGACTTTGACTCCTTCGGAAAAATTGAATGTTATCAACGCTACTTCTAATTTCCCAGGAAATTTAACGGTATCTAGTGGAGAATTAGTTGTTACGAAGGGAGCGACACTAACAGTAGGAAATATCACAGCAACATCAGGACGAGTAACTTTAGGATCAGGGGCTTCGTTATCCGCCGTTGCAGGTACTGCTGGCACTTGTACGGTGTCTAAATTAGGGATTGATTTAGAGTCCTTCCTAGTCCCTACTTATGAGACTGCAAAGTTGGGTGCGGATACAACAGTAGCGGTGAATAACAATCCTACTTTAGACCTAGTAATGGCGAATGAGACGGAGATGTATGATAATCCGCTTTTTATGAACGCTGTTACAATCCCTTTTGTGACATTGGTTTCTCTCCAAACTACTGGTGGTGTTACTACAAGTGCCGTTACTCTGAATAATGCAGATACTGCGCATTATGGGTATCAAGGATCTTGGTCTGCTGATTGGAGAAGGCCTCCTTTAGCTCCTGATCCTAGCGGCATGACACCTCTTGATAAAAGTAATACATTGTATGTGACATGGAGGCCATCCTCTAACTACGGTGTGTATAAGTTAGATCCTCAAAGAAGGGGTGAGTTGGTCCCGAATTCTTTATGGGTATCTGGATCTGCCTTAAGAACCTTTACAAATGGTTTGAAGGAACATTACGTCTCTAGAGATGTCGGATTTATTGCATCTGTACAAGCCTTAGGGGATTATGTTCTGAATTATAAGCAGGGTAACCGAGATGGCTTTCTAGCTAGGTACGGAGGTTTTCAAGCTGTTGCGGCTTCTCACTATGAAAATGGGGGGATCTTTGGGGTAGCTTTCGGTCAACTTTATGGTCAAACTAAGAGCCGTTTGTACGATTCTAAGGATGCTGGAAACATTACGATTTTGTCCTGTTTTGGACGAAGTTATATCGATGTTAAAGGAACAGAAACCGTTGTGTATTGGGAGACGGCTTATGGATATTCTGTTCATAGAATGCATACGCAGTATTTCAATGGAAAAACGAATAAGTTTGATCATTCGAAATGTCGTTGGCACAACAATAGTTATTATGCATTTGTAGGTGCAGAACATAATTTCTTGGAGTATTGTATTCCTACTCGTCAATTAGCTAGGGATTATGATCTTACAGGATTTATGCGTTTCGAAATGTCGGGAGGTTGGTCGAGTGGTGCAAAAGAAACGGGTGCTTTACCTAGACATTTTGATCGAGGAACAGGGCATAATATGTCTCTTCCAATAGGGGTTGTAGCTCATGCTGTTTCTAATGGACGAAGATCTCCTCCATCTAAATTGACGATTAACATGGGATATAGACCAGACATTTGGCGGGTGACTCCACATTGCAATATGAAAATTATTGCAAACGGAGTTAAGACTCCTATACAGGGATCTCCTCTAGCTCGGCACGCCTTCTTTTTAGAAGTTCATGATACTCTGTATGTTCGTCATTTGGGCAGAGCCTATATGAATTATTCTTTAGATGCTCGTCATCGACAAACTACGCATTTCGTATCTTTAGGATTGAATCGTATCTTTTAA PmpHMPFSLRSTSFCFLACLCSYSYGLASSPQVLTPNVIIPFKGDDIYLNGDCVFASIYAGAEQGSIISANGQNL50 PMPH_CHLMUTIVGQNHTLSFTDSQGPALQNCAFISAEEKISLRDFSSLLFSKNVSCGEKGMISGKTVSISGGDSIVFKDNSVGYSSLPSVGQTPTTPIVGDVLKGSIFCVETGLEISGVKKELVFDNTAGNFGAVFCSRAAQGDTTFTVKDCKGKILFQDNVGSCGGGVIYKGEVLFQDNEGEMLFRGNSAHDDLGILDANPQPPTEVGGGGGVICTPEKTVTFKGNKGPITFDYNFAKGRGGAIQSQTFSLVADSAVVFSNNTAEKGGGAIYALEVNVSTNGGSILFEGNRASEGGAICVSEPIAANNGGLTLHAADGDIIFSKNMTSDRPGERSAIRILDSGTNVSLNASGASKMIFYDPVVQNNPATPPTGTSGEIKINESGSGSVVFTAETLTPSEKLNVINATSNFPGNLTVSSGELVVTKGATLTVGNITATSGRVTLGSGASLSAVAGTAGTCTVSKLGIDLESFLVPTYETAKLGADTTVAVNNNPTLDLVMANETEMYDNPLFMNAVTIPFVTLVSLQTTGGVTTSAVTLNNADTAHYGYQGSWSADWRRPPLAPDPSGMTPLDKSNTLYVTWRPSSNYGVYKLDPQRRGELVPNSLWVSGSALRTFTNGLKEHYVSRDVGFIASVQALGDYVLNYKQGNRDGFLARYGGFQAVAASHYENGGIFGVAFGQLYGQTKSRLYDSKDAGNITILSCFGRSYIDVKGTETVVYWETAYGYSVHRMHTQYFNGKTNKFDHSKCRWHNNSYYAFVGAEHNFLEYCIPTRQLARDYDLTGFMRFEMSGGWSSGAKETGALPRHFDRGTGHNMSLPIGVVAHAVSNGRRSPPSKLTINMGYRPDIWRVTPHCNMKIIANGVKTPIQGSPLARHAFFLEVHDTLYVRHLGRAYMNYSLDARHRQTTHFVSLGLNRIF PmpHCATATGAGTTCTCCTCAGGTACTGACCCCGAATGTAATCATCCCTTTTAAAGGAGACGATATCTATTTAAA51 PmpH_EcOpt_dSigTGGGGATTGCGTTTTTGCAAGTATCTATGCAGGAGCAGAGCAGGGATCGATTATTTCTGCTAATGGGCAAAE. coli codonATCTGACAATCGTAGGACAAAACCACACTTTATCATTTACGGATTCCCAAGGGCCAGCCCTGCAAAATTGToptimized,GCTTTCATTTCAGCAGAAGAAAAGATCTCTCTGCGCGATTTTTCGAGCCTGTTGTTTTCGAAAAATGTTTCN-terminalTTGCGGGGAGAAAGGAATGATTTCAGGGAAAACCGTAAGCATTTCAGGGGGAGATAGTATTGTTTTTAAGGnuclearATAACTCTGTTGGTTATTCTTCATTACCGTCTGTGGGGCAAACTCCTACAACTCCAATTGTTGGCGATGTTlocalizationTTAAAGGGTTCCATTTTTTGTGTGGAGACAGGTTTAGAGATTTCTGGAGTCAAAAAAGAGCTGGTTTTCGAsignal removedTAACACTGCTGGGAATTTTGGGGCAGTATTCTGTAGTCGTGCCGCTCAAGGAGACACGACTTTCACAGTGAAAGACTGTAAGGGTAAAATTCTGTTTCAAGATAACGTAGGCTCTTGTGGAGGCGGCGTAATTTATAAAGGGGAAGTACTGTTCCAAGATAATGAAGGAGAAATGCTGTTCCGCGGAAATTCAGCTCATGATGATTTGGGAATTCTGGATGCTAACCCACAGCCTCCTACTGAAGTAGGAGGTGGGGGTGGTGTCATTTGTACCCCAGAGAAAACGGTAACTTTTAAGGGGAATAAAGGGCCTATTACCTTTGATTATAATTTTGCAAAAGGTCGCGGAGGGGCAATCCAATCACAGACCTTTTCTTTGGTAGCTGATAGTGCTGTTGTTTTCAGTAATAATACAGCTGAGAAAGGTGGAGGCGCCATTTATGCTCTGGAGGTTAACGTGAGCACAAATGGAGGATCTATTCTGTTTGAGGGAAATCGCGCTTCTGAGGGTGGGGCTATCTGTGTGAGCGAGCCGATCGCTGCTAATAATGGAGGGCTGACTTTACATGCTGCTGATGGGGACATTATTTTCTCGAAAAATATGACGAGTGATCGTCCTGGAGAACGCAGTGCAATCCGGATCTTAGATAGTGGAACAAATGTCTCTTTAAATGCTTCAGGGGCATCGAAGATGATTTTTTATGATCCTGTTGTGCAAAATAATCCGGCAACTCCACCTACTGGTACGTCTGGGGAAATTAAGATCAATGAGTCCGGGAGTGGATCGGTTGTGTTTACAGCAGAGACTTTGACTCCTTCGGAAAAATTGAATGTTATCAACGCTACTTCTAATTTCCCAGGAAATTTAACGGTATCTAGTGGAGAGCTCGTTGTTACGAAGGGAGCGACACTGACAGTAGGAAATATCACAGCAACATCAGGACGCGTAACTTTAGGATCAGGGGCTTCGTTATCCGCCGTTGCAGGTACTGCTGGCACTTGTACGGTGTCTAAATTAGGGATTGATTTAGAGTCCTTCCTGGTCCCTACTTATGAGACTGCAAAGTTGGGTGCGGATACAACAGTAGCGGTGAATAACAATCCTACTTTAGACCTGGTAATGGCGAATGAGACGGAGATGTATGATAATCCGCTGTTTATGAACGCTGTTACAATCCCTTTTGTGACATTGGTTTCTCTGCAAACTACTGGTGGTGTTACTACAAGTGCCGTTACTCTGAATAATGCAGATACTGCGCATTATGGGTATCAAGGATCTTGGTCTGCTGATTGGCGCCGCCCTCCTTTAGCTCCTGATCCTAGCGGCATGACACCTCTGGATAAAAGTAATACATTGTATGTGACATGGCGCCCATCCTCTAACTACGGTGTGTATAAGTTAGATCCCATGGCCCGGCGTGGTGAGTTGGTCCCGAATTCTTTATGGGTATCTGGATCTGCCTTACGCACCTTTACAAATGGTTTGAAGGAACATTACGTCTCTCGCGATGTCGGATTTATTGCATCTGTACAAGCCTTAGGGGATTATGTTCTGAATTATAAGCAGGGTAACCGCGATGGCTTTCTGGCTCGCTACGGAGGTTTTCAAGCTGTTGCGGCTTCTCACTATGAAAATGGGGGGATCTTTGGGGTAGCTTTCGGTCAACTGTATGGTCAAACTAAGAGCCGTTTGTACGATTCTAAGGATGCTGGAAACATTACGATTTTGTCCTGTTTTGGACGCAGTTATATCGATGTTAAAGGAACAGAAACCGTTGTGTATTGGGAGACGGCTTATGGATATTCTGTTCATCGCATGCATACGCAGTATTTCAATGGAAAAACGAATAAGTTTGATCATTCGAAATGTCGTTGGCACAACAATAGTTATTATGCATTTGTAGGTGCAGAACATAATTTCTTGGAGTATTGTATTCCTACTCGTCAATTAGCTCGCGATTATGATCTGACAGGATTTATGCGTTTCGAAATGTCGGGAGGTTGGTCGAGTGGTGCAAAAGAAACGGGTGCTTTACCTCGCCATTTTGATCGCGGAACAGGGCATAATATGTCTCTGCCAATTGGGGTTGTAGCTCATGCTGTTTCTAATGGACGCCGCTCTCCTCCATCTAAATTGACGATTAACATGGGATATCGCCCAGACATTTGGCGGGTGACTCCACATTGCAATATGAAAATTATTGCAAACGGAGTTAAGACTCCTATTCAGGGATCTCCTCTGGCTCGGCACGCCTTCTTTTTAGAAGTTCATGATACTCTGTATGTTCGTCATTTGGGCCGCGCCTATATGAATTATTCTTTAGATGCTCGTCATCGCCAAACTACGCATTTCGTATCTTTAGGATTGAATCGTATCTTTtaaGGATCC PmpHMSSPQVLTPNVIIPFKGDDIYLNGDCVFASIYAGAEQGSIISANGQNLTIVGQNHTLSFTDSQGPALQNCA52 PmpH_EcOpt_dSigFISAEEKISLRDFSSLLFSKNVSCGEKGMISGKTVSISGGDSIVFKDNSVGYSSLPSVGQTPTTPIVGDVLE. coli codonKGSIFCVETGLEISGVKKELVFDNTAGNFGAVFCSRAAQGDTTFTVKDCKGKILFQDNVGSCGGGVIYKGEoptimized,VLFQDNEGEMLFRGNSAHDDLGILDANPQPPTEVGGGGGVICTPEKTVTFKGNKGPITFDYNFAKGRGGAIN-terminalQSQTFSLVADSAVVFSNNTAEKGGGAIYALEVNVSTNGGSILFEGNRASEGGAICVSEPIAANNGGLTLHAnuclearADGDIIFSKNMTSDRPGERSAIRILDSGTNVSLNASGASKMIFYDPVVQNNPATPPTGTSGEIKINESGSGlocalizationSVVFTAETLTPSEKLNVINATSNFPGNLTVSSGELVVTKGATLTVGNITATSGRVTLGSGASLSAVAGTAGsignal removedTCTVSKLGIDLESFLVPTYETAKLGADTTVAVNNNPTLDLVMANETEMYDNPLFMNAVTIPFVTLVSLQTTGGVTTSAVTLNNADTAHYGYQGSWSADWRRPPLAPDPSGMTPLDKSNTLYVTWRPSSNYGVYKLDPMARRGELVPNSLWVSGSALRTFTNGLKEHYVSRDVGFIASVQALGDYVLNYKQGNRDGFLARYGGFQAVAASHYENGGIFGVAFGQLYGQTKSRLYDSKDAGNITILSCFGRSYIDVKGTETVVYWETAYGYSVHRMHTQYFNGKTNKFDHSKCRWHNNSYYAFVGAEHNFLEYCIPTRQLARDYDLTGFMRFEMSGGWSSGAKETGALPRHFDRGTGHNMSLPIGVVAHAVSNGRRSPPSKLTINMGYRPDIWRVTPHCNMKIIANGVKTPIQGSPLARHAFFLEVHDTLYVRHLGRAYMNYSLDARHRQTTHFVSLGLNRIF PmpHCATATGAGTTCTCCTCAGGTACTGACCCCGAATGTAATCATCCCTTTTAAAGGAGACGATATCTATTTAAA53 PmpH_EcOpt_dSig_TGGGGATTGCGTTTTTGCAAGTATCTATGCAGGAGCAGAGCAGGGATCGATTATTTCTGCTAATGGGCAAAdPMPATCTGACAATCGTAGGACAAAACCACACTTTATCATTTACGGATTCCCAAGGGCCAGCCCTGCAAAATTGTE. coli codonGCTTTCATTTCAGCAGAAGAAAAGATCTCTCTGCGCGATTTTTCGAGCCTGTTGTTTTCGAAAAATGTTTCoptimized,TTGCGGGGAGAAAGGAATGATTTCAGGGAAAACCGTAAGCATTTCAGGGGGAGATAGTATTGTTTTTAAGGN-terminalATAACTCTGTTGGTTATTCTTCATTACCGTCTGTGGGGCAAACTCCTACAACTCCAATTGTTGGCGATGTTnuclearTTAAAGGGTTCCATTTTTTGTGTGGAGACAGGTTTAGAGATTTCTGGAGTCAAAAAAGAGCTGGTTTTCGAlocalizationTAACACTGCTGGGAATTTTGGGGCAGTATTCTGTAGTCGTGCCGCTCAAGGAGACACGACTTTCACAGTGAsignal removed,AAGACTGTAAGGGTAAAATTCTGTTTCAAGATAACGTAGGCTCTTGTGGAGGCGGCGTAATTTATAAAGGGadhesion domainGAAGTACTGTTCCAAGATAATGAAGGAGAAATGCTGTTCCGCGGAAATTCAGCTCATGATGATTTGGGAAT(ChlamPMP_M)TCTGGATGCTAACCCACAGCCTCCTACTGAAGTAGGAGGTGGGGGTGGTGTCATTTGTACCCCAGAGAAAAremovedCGGTAACTTTTAAGGGGAATAAAGGGCCTATTACCTTTGATTATAATTTTGCAAAAGGTCGCGGAGGGGCAATCCAATCACAGACCTTTTCTTTGGTAGCTGATAGTGCTGTTGTTTTCAGTAATAATACAGCTGAGAAAGGTGGAGGCGCCATTTATGCTCTGGAGGTTAACGTGAGCACAAATGGAGGATCTATTCTGTTTGAGGGAAATCGCGCTTCTGAGGGTGGGGCTATCTGTGTGAGCGAGCCGATCGCTGCTAATAATGGAGGGCTGACTTTACATGCTGCTGATGGGGACATTATTTTCTCGAAAAATATGACGAGTGATCGTCCTGGAGAACGCAGTGCAATCCGGATCTTAGATAGTGGAACAAATGTCTCTTTAAATGCTTCAGGGGCATCGAAGATGATTTTTTATGATCCTGTTGTGCAAAATAATCCGGCAACTCCACCTACTGGTACGTCTGGGGAAATTAAGATCAATGAGTCCGGGAGTGGATCGGTTGTGTTTACAGCAGAGACTTTGACTCCTTCGGAAAAATTGAATGTTATCAACGCTACTTCTAATTTCCCAGGAAATTTAACGGTATCTAGTGGAGAGCTCTCCTCTAACTACGGTGTGTATAAGTTAGATCCCATGGCCCGGCGTGGTGAGTTGGTCCCGAATTCTTTATGGGTATCTGGATCTGCCTTACGCACCTTTACAAATGGTTTGAAGGAACATTACGTCTCTCGCGATGTCGGATTTATTGCATCTGTACAAGCCTTAGGGGATTATGTTCTGAATTATAAGCAGGGTAACCGCGATGGCTTTCTGGCTCGCTACGGAGGTTTTCAAGCTGTTGCGGCTTCTCACTATGAAAATGGGGGGATCTTTGGGGTAGCTTTCGGTCAACTGTATGGTCAAACTAAGAGCCGTTTGTACGATTCTAAGGATGCTGGAAACATTACGATTTTGTCCTGTTTTGGACGCAGTTATATCGATGTTAAAGGAACAGAAACCGTTGTGTATTGGGAGACGGCTTATGGATATTCTGTTCATCGCATGCATACGCAGTATTTCAATGGAAAAACGAATAAGTTTGATCATTCGAAATGTCGTTGGCACAACAATAGTTATTATGCATTTGTAGGTGCAGAACATAATTTCTTGGAGTATTGTATTCCTACTCGTCAATTAGCTCGCGATTATGATCTGACAGGATTTATGCGTTTCGAAATGTCGGGAGGTTGGTCGAGTGGTGCAAAAGAAACGGGTGCTTTACCTCGCCATTTTGATCGCGGAACAGGGCATAATATGTCTCTGCCAATTGGGGTTGTAGCTCATGCTGTTTCTAATGGACGCCGCTCTCCTCCATCTAAATTGACGATTAACATGGGATATCGCCCAGACATTTGGCGGGTGACTCCACATTGCAATATGAAAATTATTGCAAACGGAGTTAAGACTCCTATTCAGGGATCTCCTCTGGCTCGGCACGCCTTCTTTTTAGAAGTTCATGATACTCTGTATGTTCGTCATTTGGGCCGCGCCTATATGAATTATTCTTTAGATGCTCGTCATCGCCAAACTACGCATTTCGTATCTTTAGGATTGAATCGTATCTTTtaaGGATCC PmpHMSSPQVLTPNVIIPFKGDDIYLNGDCVFASIYAGAEQGSIISANGQNLTIVGQNHTLSFTDSQGPALQNCA54 PmpH_EcOpt_dSig_FISAEEKISLRDFSSLLFSKNVSCGEKGMISGKTVSISGGDSIVFKDNSVGYSSLPSVGQTPTTPIVGDVLdPMPKGSIFCVETGLEISGVKKELVFDNTAGNFGAVFCSRAAQGDTTFTVKDCKGKILFQDNVGSCGGGVIYKGEE. coli codonVLFQDNEGEMLFRGNSAHDDLGILDANPQPPTEVGGGGGVICTPEKTVTFKGNKGPITFDYNFAKGRGGAIoptimized,QSQTFSLVADSAVVFSNNTAEKGGGAIYALEVNVSTNGGSILFEGNRASEGGAICVSEPIAANNGGLTLHAN-terminalADGDIIFSKNMTSDRPGERSAIRILDSGTNVSLNASGASKMIFYDPVVQNNPATPPTGTSGEIKINESGSGnuclearSVVFTAETLTPSEKLNVINATSNFPGNLTVSSGELSSNYGVYKLDPMARRGELVPNSLWVSGSALRTFTNGlocalizationLKEHYVSRDVGFIASVQALGDYVLNYKQGNRDGFLARYGGFQAVAASHYENGGIFGVAFGQLYGQTKSRLYsignal removed,DSKDAGNITILSCFGRSYIDVKGTETVVYWETAYGYSVHRMHTQYFNGKTNKFDHSKCRWHNNSTYAFVGAadhesion domainEHNFLEYCIPTRQLARDYDLTGFMRFEMSGGWSSGAKETGALPRHFDRGTGHNMSLPIGVVAHAVSNGRRS(ChlamPMP_M)PPSKLTINMGYRPDIWRVTPHCNMKIIANGVKTPIQGSPLARHAFFLEVHDTLYVRHLGRAYMNYSLDARHremoved RQTTHFVSLGLNRIF

In a first set of experiments, codon optimization was used to altersequences for Pmp-tNLP expression in E. coli cell-free lysates. Codonoptimization of the Δ49ApoA1 or ApoE4 and Pmp sequences resulted inproduction of full-length protein with and without adhesion domain.Co-translation reaction conditions using plasmids encoding Δ49ApoA1 orApoE4 and Pmp were initially screened using a bodipy-lysine fluorescentamino acid to simplify visualization of protein expression andsolubility screening.

The bodipy-lysine fluorescent amino acid is randomly inserted at lysinepositions within the protein at a low insertion rate. Pmp expression wasobserved in the cell-free reactions include both DMPC lipid andtelodendrimer PEG^(5k)-CA₈. After the cell-free reaction was completed,the total cell-free mixtures were resolved by SDS-PAGE and imaged byfluorescent imaging. The plasmids encoding Pmp and scaffolding proteinApoA1 or ApoE4 is at ratio of 50:1 (FIG. 21A).

The results illustrated in FIG. 21A show SDS-PAGE images ofcodon-optimized fluorescent labeled Pmps (PmpC, PmpE, PmpF, PmpG, andPmpH) and truncated versions that lack the adhesion domain. Theseresults show that full-length and truncation Pmp proteins can becell-free expressed in the presence of lipid and telodendrimer as willbe understood by a skilled person.

In a second set of experiments, co-translation reaction using plasmidsencoding Δ49ApoA1 or ApoE4 and PmpH were set up in the presence ofbodipy-lysine fluorescent amino acid, DMPC lipid and telodendrimerPEG^(5k)-CA₈. The plasmids encoding Pmp and scaffolding protein ApoA1 orApoE4 is at ratio of 50:1. Reactions were scaled up to 1 mL to producesufficient quantities of Pmp for subsequent nickel purificationutilizing the HIS tag on the apolipoprotein scaffold component of thetNLP.

The purification provided a complex that was >95% pure based on SDS-PAGEanalysis. On average, a 1 mL reaction produced ˜200 μg of PmpH (FIG.21B) based on gel densitometry. Distinct bands indicated that the twoproteins, apolipoprotein and Pmp, were co-purifying as a complex.

The results are illustrated in FIG. 21B which shows SDS-PAGE images ofcell-free expressed and nickel-purified PmpH and Δ49 ApoA₁, a truncatedversion of mouse ApoA1 in which the N-terminal 49 amino acids wereremoved. A 1 mL reaction produced ˜200 μg of PmpH. Although in this setof experiments MOMP was not cotranslated with PmpH, MOMP can betranslated with PmpH as will be understood by a skilled person uponreading of the present disclosure.

Example 11: Structural and Protective Assessment of Chlamydial Proteins

This example further demonstrates that NLPs are a vaccine deliveryplatform for membrane protein antigens. In addition, the example alsoconfirms that C. muridarum MOMP is amenable to gene optimization,cell-free expression, and purification in the NLP complex.

The experiments were carried out using the approaches previouslydescribed in Examples 4 and 5. In particular, an Escherichia coli-basedcell-free system was used to express a MOMP protein from themouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). Thecodon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1(Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal49 amino acids were removed. This co-translation process produced mMOMPsupported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP).The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar tothe native MOMP protein. This cell-free process produced on average 1.5mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-freereaction.

FIGS. 22A-22C demonstrates a cell-free production of MOMP co-translatedwith ApoA₁ Δ49A₁. FIG. 22A shows SDS-PAGE images of cell-free expressedand purified MOMP and ApoA1 Δ49A1.

FIG. 22B shows exemplary results of dot blot analysis MOMP-NLP and NLPassemblies treated with heat and reducing agent. The MOMP-NLP and NLPassemblies were blotted in triplicate and probed with mAb40, mAbHIS andmAb18b. The confirmation of the MOMP trimer was confirmed using theconformational monoclonal antibody mAb18b. Addition of heat and DTTresults in a decrease in signal as detected by mAb18b, indicating a lossin trimer formation. mAb40 recognizes both MOMP monomer and trimer as itbinds to a linear epitope. The mAbHIS recognizes the HIS tag on theΔ49A1 protein.

FIG. 22C shows conductance traces recorded at 50 mV applied voltage inphysiological conditions after NLP alone and MOMP-NLP were added to themeasurement chamber. Current increases observed after MOMP-NLP additionindicate the formation of bilayer pore formation by MOMP proteins,indicating functional MOMP insertion.

Using the mMOMP-tNLP formulation, a unique approach is demonstrated tosolubilizing and administering membrane-bound proteins for futurevaccine development. This method can also be applied to include otherantigens such as Pmps while maintaining their full functionality andimmunogenicity.

Example 12: Structural and Protective Assessment of Chlamydial Proteins

The experiments in this example were carried out using proceduresdescribed in Example 7.

In particular, the protective response of MOMP-NLP was evaluated in amouse intranasal challenge study. Briefly, were inoculated intranasallywith formulated controls (PBS or empty NLPs) or different formulationsof MOMP-NLPs. With chlamydial challenges, the mice undergo weight lossand recovery. The recovery is an indication of protection for anyformulation.

In FIG. 24, weight loss over time following intranasal (i.n) challengewith C. muridarum was used as a measure of protection. Data was analyzedusing RM two-way ANOVA with Sidak's multiple comparison analysis.

In vivo vaccination with MOMP-NLPs displayed strong protection againstChlamydia challenge in mice compared to empty NLPs and PBS control.Additionally, mice immunized with MOMP:NLP lost significant body weightby 4 days post challenge (d.p.c.) but by 10 d.p.c. have recovered someof their weight (FIG. 24). The positive control, Chlamydia elementarybody (EB), demonstrates complete protection.

These combined preliminary results demonstrate the feasibility ofextending NLP approach to the genital model for further vaccinedevelopment.

Additionally, since using systemic and/or mucosal routes forimmunization, a better protection has been observed when using bothroutes. It is therefore expected that delivery of MOMP-NLPs by bothroutes will result in enhancing systemic and mucosal humoral andcellular memory immune responses.

In summary, described herein is a telodendrimer-nanolipoprotein particle(t-NLP), comprising one or more membrane forming lipids, one or moretelodendrimers, and a scaffold protein and a Chlamydia major outermembrane protein (MOMP) comprising a MOMP hydrophobic region, andrelated compositions methods and systems.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the materials, compositions, systems andmethods of the disclosure, and are not intended to limit the scope ofwhat the inventors regard as their disclosure. Those skilled in the artwill recognize how to adapt the features of the exemplified NLPs andrelated uses to additional NLPs formed by other cationic lipids,membrane forming lipids, scaffold proteins, additives, and possiblyfunctionalized amphipathic compounds and membrane proteins according tovarious embodiments and scope of the claims.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe disclosure pertains.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Summary, Detailed Description, andExamples is hereby incorporated herein by reference. All referencescited in this disclosure are incorporated by reference to the sameextent as if each reference had been incorporated by reference in itsentirety individually. However, if any inconsistency arises between acited reference and the present disclosure, the present disclosure takesprecedence. Further, the computer readable form of the sequence listingof the ASCII text file IL13105-PCT-Seq-List-ST25 is incorporated hereinby reference in its entirety.

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe disclosure claimed. Thus, it should be understood that although thedisclosure has been specifically disclosed by embodiments, exemplaryembodiments and optional features, modification and variation of theconcepts herein disclosed can be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this disclosure as defined by the appended claims.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. As used in this specification and the appended claims,the singular forms “a,” “an,” and “the” include plural referents unlessthe content clearly dictates otherwise. The term “plurality” includestwo or more referents unless the content clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the disclosure pertains.

When a Markush group or other grouping is used herein, all individualmembers of the group and all combinations and possible subcombinationsof the group are intended to be individually included in the disclosure.Every combination of components or materials described or exemplifiedherein can be used to practice the disclosure, unless otherwise stated.One of ordinary skill in the art will appreciate that methods, deviceelements, and materials other than those specifically exemplified may beemployed in the practice of the disclosure without resort to undueexperimentation. All art-known functional equivalents, of any suchmethods, device elements, and materials are intended to be included inthis disclosure. Whenever a range is given in the specification, forexample, a temperature range, a frequency range, a time range, or acomposition range, all intermediate ranges and all subranges, as wellas, all individual values included in the ranges given are intended tobe included in the disclosure. Any one or more individual members of arange or group disclosed herein may be excluded from a claim of thisdisclosure. The disclosure illustratively described herein suitably maybe practiced in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein.

A number of embodiments of the disclosure have been described. Thespecific embodiments provided herein are examples of useful embodimentsof the invention and it will be apparent to one skilled in the art thatthe disclosure can be carried out using a large number of variations ofthe devices, device components, methods steps set forth in the presentdescription. As will be obvious to one of skill in the art, methods anddevices useful for the present methods may include a large number ofoptional composition and processing elements and steps.

In particular, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

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1. A telodendrimer-nanolipoprotein particle (t-NLP), comprising one ormore membrane forming lipids, one or more telodendrimers, one or moreadjuvants, and a scaffold protein and a Chlamydia major outer membraneprotein (MOMP) and/or a fragment thereof comprising a MOMP hydrophobicregion, wherein the one or more membrane forming lipids are arranged ina discoidal membrane lipid bilayer stabilized by the scaffold proteinand the one or more telodendrimers, with the membrane lipid bilayerattaching the MOMP and/or the fragment thereof through interaction ofthe hydrophobic region with the membrane lipid bilayer and the one ormore adjuvants through binding of the one or more adjuvants to thediscoidal membrane bilayer or to a component thereof.
 2. Thetelodendrimer-nanolipoprotein particle of claim 1, having a size between5 nm to 100 nm in diameter and comprising a telodendrimer to lipid ratioof 1:10 to 1:1000 a ratio of scaffold protein to lipid of 1:30 to 1:100and a ratio of MOMP to scaffold protein of 20:1 to 1:4.
 3. (canceled) 4.(canceled)
 5. (canceled)
 6. (canceled)
 7. (canceled)
 8. (canceled) 9.(canceled)
 10. (canceled)
 11. (canceled)
 12. Thetelodendrimer-nanolipoprotein particle of claim 1, wherein thetelodendrimer is a compound of formula (I):(T)_(m)-(A)_(p)-L-D-(R)_(n)  (I) wherein D is a dendrimer T is a tailgroup; A is a spacer moiety configured to be directly covalentlyconnected to each T and to a linker moiety L, and comprises a polymer of1 to m number of spacer A monomers, wherein the spacer A monomercomprises a substituted or unsubstituted linear C1-C15 alkyl; branchedC3-C15 alkyl; cyclic C3-C15 alkyl; linear, cyclic, or branched C2-C15alkenyl; linear, cyclic, or branched C2-C15 alkynyl; C6-C20 substitutedor unsubstituted aryl; and C6-C20 substituted or unsubstitutedheteroaryl, R can be a detergent moiety, a lipid and/or an amino acid mis 0-20 and p is 0-1, and wherein m is 0 or 1 when p is 0; or m is 2-20when p is
 1. 13. (canceled)
 14. (canceled)
 15. (canceled)
 16. Thetelodendrimer-nanolipoprotein particle of claim 1, wherein the MOMP is aMOMP of Chlamydia species Chlamydia trachomatis, Chlamydia pneumoniae,and Chlamydia psittaci (human pathogens), Chlamydia suis (affects onlyswine), Chlamydia pecorum (affects cows/swine/koala) and Chlamydiapneumonia (affects koala) and Chlamydia muridarum (affects only mice andhamsters) or a variant thereof or a fragment thereof, and wherein thevariant maintains a beta barrel structure with 18 transmembrane regionsseparated by loops of the MOMP.
 17. (canceled)
 18. Thetelodendrimer-nanolipoprotein particle of claim 1, wherein the MOMP is aMOMP immunogenic fragment.
 19. The telodendrimer-nanolipoproteinparticle of claim 1, wherein the membrane forming lipid comprises atleast one phospholipid, selected from soy phosphatidylcholine, eggphosphatidylcholine, soy phosphatidylglycerol, egg phosphatidylglycerol,palmitoyl-oleoyl-phosphatidylcholine distearoylphosphatidylcholine,distearoylphosphatidylglycerol phosphatidylcholine,phosphatidylglycerol, sphingomyelin, phosphatidylserine, phosphatidicacid, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylinositol, cephalin,cardiolipin, cerebrosides, dicetylphosphate,dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine,dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol,stearoyl-palmitoyl-phosphatidylcholine,di-palmitoyl-phosphatidylethanolamine,distearoyl-phosphatidylethanolamine, di-myrstoyl-phosphatidylserine,di-myrstoyl-phosphatidylcholine and dioleyl-phosphatidylcholine. 20.(canceled)
 21. The telodendrimer-nanolipoprotein particle of claim 1,wherein the scaffold protein is one or more of a human derived apoE4, atruncated version of human derived apoE4, a human derived apoE3, atruncated version of human derived apoE3, a human derived apoE2, atruncated version of human derived apoE2, a human derived apoA1, atruncated version of human derived apoA1, a mouse derived apoE4, atruncated version of mouse derived apoE4, mouse derived apoE3, truncatedversions of mouse derived apoE3, a mouse derived apoE2, a truncatedversion of mouse derived apoE2, a mouse derived apoA1, a truncatedversion of mouse derived apoA1, a rat derived apoE4, a truncated versionof rat derived apoE4, a rat derived apoE3, a truncated version of ratderived apoE3, a rat derived apoE2, a truncated version of rat derivedapoE2, a rat derived apoA1, a truncated version of rat derived apoA1, alipophorin, a synthetic cyclic peptide mimicking an apolipoproteinfunction.
 22. (canceled)
 23. (canceled)
 24. (canceled)
 25. (canceled)26. The telodendrimer-nanolipoprotein particle of claim 1, furthercomprising one or more polymorphic membrane proteins.
 27. Thetelodendrimer-nanolipoprotein particle of claim 26, the one or morepolymorphic membrane proteins are selected from Pmp A, PmpB, PmpC, PmpD,PmpE, PmpF, PmpG, PmpH, and PmpI from Chlamydia muridarum or C.trachomatis.
 28. (canceled)
 29. (canceled)
 30. (canceled)
 31. (canceled)32. (canceled)
 33. A method to provide a telodendrimer-nanolipoproteinparticle presenting a Chlamydia major outer membrane proteins (MOMP),the method comprising providing one or more membrane forming lipids, oneor more telodendrimers, one or more adjuvants, a polynucleotide codingfor the MOMP and a polynucleotide coding for a scaffold protein; mixingthe one or more membrane forming lipids and the one or moretelodendrimers to provide a lipid-telodendrimer mixture; mixinglipid-telodendrimer mixture with, the polynucleotide coding for the MOMPand the polynucleotide coding for the scaffold protein with an in vitrocell free translation system to provide a single reaction mixture;translating the polynucleotide within the single reaction mixture viathe in vitro cell free translation system, and contacting the singlereaction mixture with the one or more adjuvants wherein the mixing thetranslating and the contacting are performed to allow self-assembly ofthe scaffold protein, the one or more membrane forming lipids and theone or more telodendrimers into a nanolipoprotein particle, thenanolipoprotein particle comprising the MOMP within a discoidal membranelipid bilayer formed by the one or more membrane forming lipids andstabilized by the scaffold protein, the membrane lipid bilayer attachingthe MOMP through interaction of a hydrophobic region of the MOMP withthe membrane lipid bilayer the membrane lipid bilayer further attachingthe one or more adjuvants through binding of the one or more adjuvantsto the discoidal membrane bilayer or to a component thereof. 34.(canceled)
 35. (canceled)
 36. (canceled)
 37. (canceled)
 38. (canceled)39. (canceled)
 40. (canceled)
 41. (canceled)
 42. (canceled)
 43. A systemto provide a t-NLP comprising Chlamydia major outer membrane proteins(MOMP), the system comprising one or more membrane forming lipids, oneor more telodendrimers, one or more adjuvants, a polynucleotide codingfor Chlamydia major outer membrane proteins (MOMP) and a polynucleotidecoding for a scaffold protein for simultaneous combined or sequentialuse in the method to provide a t-NLP presenting a MOMP of claim
 33. 44.A method to provide a telodendrimer-nanolipoprotein particle presentinga Chlamydia major outer membrane proteins (MOMP), the method comprisingproviding one or more membrane forming lipids, one or moretelodendrimers, one or more adjuvants, a scaffold protein and apolynucleotide coding for the MOMP; mixing the one or more membraneforming lipids and the one or more telodendrimers to provide alipid-telodendrimer mixture; mixing lipid-telodendrimer mixture with thescaffold protein and the polynucleotide coding for the MOMP with an invitro cell free translation system to provide a single reaction mixture;translating the polynucleotide within the single reaction mixture viathe in vitro cell free translation system, and contacting the singlereaction mixture with the one or more adjuvants wherein the mixing thetranslating and the contacting are performed to allow self-assembly ofthe scaffold protein, the one or more membrane forming lipids and theone or more telodendrimers into a nanolipoprotein particle, thenanolipoprotein particle comprising the MOMP within a discoidal membranelipid bilayer formed by the one or more membrane forming lipids andstabilized by the scaffold protein, the membrane lipid bilayer attachingthe MOMP through interaction of a hydrophobic region of the MOMP withthe membrane lipid bilayer, the membrane lipid bilayer further attachingthe one or more adjuvants through binding of the one or more adjuvantsto the membrane lipid bilayer and/or to a component thereof.
 45. Asystem to provide a t-NLP comprising Chlamydia major outer membraneproteins (MOMP), the system comprising one or more membrane forminglipids, one or more telodendrimers, one or more adjuvants apolynucleotide coding for Chlamydia major outer membrane proteins (MOMP)and a polynucleotide coding for a scaffold protein for simultaneouscombined or sequential use in the method to provide a t-NLP presenting aMOMP of claim
 44. 46. A composition comprising one or moretelodendrimer-nanolipoprotein particles of claim 1 (MOMP-t-NLPs)together with a suitable vehicle.
 47. (canceled)
 48. (canceled) 49.(canceled)
 50. The composition of claim 46, wherein the composition isformulated for systemic administration, which includes parenteraladministration and more particularly intravenous, intradermic, andintramuscular administration.
 51. The composition of claim 46, whereinthe composition is formulated for non-parenteral administration.
 52. Thecomposition of claim 46, wherein the composition is formulated forintranasal, intratracheal, vaginal, oral, and sublingual administration.53. A method for immunizing an individual against Chlamydia, the methodcomprising administering to the individual an effective amount one ormore telodendrimer-nanolipoprotein particles (MOMP-t-NLPs) of claim 1for a time and under conditions to allow contact of the MOMP-t-NLP withthe immunitary system of the individual.
 54. The method of claim 53,wherein the administering is performed via intranasal, intramuscular ora combination of intranasal and/or intramuscular route.
 55. The methodof claim 53, wherein effective amount of MOMP-t-NLP is from 1 to 20 ug.56. A method for treating or preventing a Chlamydia infection orconditions associated thereto in an individual, the method comprisingadministering to the individual a one or moretelodendrimer-nanolipoprotein particle of claim 1 (MOMP-t-NLPs) in aneffective amount to elicit an immunitary response to the MOMP-t-NLPs inthe individual.
 57. The method of claim 56, wherein the administering isperformed via intranasal or intramuscular route or a combination ofintranasal and intramuscular routes.
 58. The method of claim 56, whereinthe effective amount of MOMP-tNLP ranges are from 1 to 20 ug. 59.(canceled)
 60. (canceled)
 61. (canceled)
 62. A system for treating orpreventing a Chlamydia infection or conditions associated thereto in anindividual, the system comprising one or moretelodendrimer-nanolipoprotein particles of claim 1 together with one ormore adjuvant or adjuvant-NLPs for simultaneous, combined or sequentialuse method for treating or preventing a Chlamydia infection orconditions associated thereto in an individual, the method comprisingadministering to the individual a one or moretelodendrimer-nanolipoprotein particle of claim 1 (MOMP-t-NLPs) in aneffective amount to elicit an immunitary response to the MOMP-t-NLPs inthe individual.
 63. The telodendrimer-nanolipoprotein particle of claim1, wherein at least one adjuvant of the one or more adjuvants comprisesa hydrophobic region and the at least one adjuvant of the one or moreadjuvants is attached to the membrane lipids bilayer through interactionof the hydrophobic region with the membrane lipid bilayer.
 64. Thetelodendrimer-nanolipoprotein particle of claim 1, wherein thetelodendrimer nanolipoprotein particle further comprises afunctionalized lipid having a hydrophobic portion and a hydrophilicportion in a configuration where the hydrophobic portion anchor isattached to the membrane lipid bilayer of the telodendrimernanolipoprotein particle and the hydrophilic portion is presented on themembrane lipid bilayer, the hydrophilic portion comprising an anchorsubstrate functional group, and wherein at least one adjuvant of the oneor more adjuvants comprises an anchor functional group, the at least oneadjuvant attached to the membrane lipid bilayer through binding of theat anchor functional group to the anchor substrate functional group ofthe functionalized lipid.
 65. The telodendrimer-nanolipoprotein particleof claim 1, wherein the one or more adjuvants comprise CpG, FSL1, LPSand/or or MPLA.
 66. The telodendrimer-nanolipoprotein particle of claim1, wherein the telodendrimer-nanolipoprotein particle comprises two ormore adjuvants.
 67. The telodendrimer-nanolipoprotein particle of claim1, wherein the two adjuvants or more adjuvants comprise CpG and FSL1.68. The composition of claim 46, wherein the composition comprisestelodendrimer-nanolipoprotein particle of claim 1 comprising two or moreadjuvants.
 69. The method of claim 53, wherein the one or moretelodendrimer-nanolipoprotein particles (MOMP-t-NLPs) comprises same ordifferent two or more adjuvants.